Pygopus in diagnosis and treatment of cancer

ABSTRACT

Expression of pygopus mRNA and Pygopus protein in established cancer cell lines and in patient tumors is described. Pygopus is shown to be a feasible diagnostic and prognostic indicator. Pygopus is also useful in cancer treatment therapy, for example in disrupting strategies that specifically target the activity of pygopus in cancer cells.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/463,309, filed Apr. 17, 2003, and U.S. Provisional Application No.60/496,012, filed Aug. 19, 2003, the contents of which are hereinincorporated by reference.

FIELD OF INVENTION

The present invention relates to the Pygopus gene and its use in thediagnosis and treatment of cancer.

BACKGROUND OF THE INVENTION

Reliable markers for malignant tumor differentiation are necessary foraccurate diagnosis of cancer. In addition, many of these markers mayprove useful as targets for therapy, especially if they are required forgrowth or metastasis of cancer cells. Present therapies for cancerinclude surgery, radiation and chemotherapy. For the most part,chemotherapy involves the use of general DNA synthesis inhibitors, DNAstructure disruptors and DNA adducts in which chemical agents are addedto the primary DNA molecule. The problem with these approaches is thatthey are not specific to cancer cells, so there are many seriousnegative side effects associated with chemotherapy such as nausea, hairloss, gastrointestinal disorders and disruption of normal brainfunction.

The Wnt-signaling pathway has been known for some time to be abnormallyactivated in colon cancer. Pygopus is a downstream effector of thispathway. The problem we faced was to determine how to use human Pygopusin cancer diagnostic and therapy.

The canonical Wnt/β-catenin pathway is normally required for embryonicdevelopment and for the controlled proliferation of adult stem cells.The critical mediator of the pathway is β-catenin, a multifunctionalprotein whose activity depends on its cytoplasmic localization. In theabsence of Wnt signaling, the majority of β-catenin is associated withthe plasma membrane, where it is required with E-cadherin for celladhesion. Cytoplasmic levels of free β-catenin are regulated by adestruction complex consisting of tumor suppressor proteins that includeGlycogen Synthase Kinase-3 beta (GSK-3β), Axin, Adenomatous PolyposisColi (APC) and Casein Kinase I which cooperatively direct cytoplasmicβ-catenin for proteosome-mediated degradation. The binding of Wnt to itsreceptor relieves degradation of cytosolic β-catenin, allowing it toaccumulate in the nucleus where it assembles into a complex that bindsto genes involved in cell cycle progression. This complex is composed ofLeukemia Enhancer/T-cell Factor (LEF)/TCF-1, B-cell lymphoma-9 protein(BCL-9) and the nuclear protein, Pygopus. Localized chromatin remodelingeffects by Pygopus, specifically bound to BCL-9 are thought to allowaccess of the basal transcriptional machinery to initiate target genetranscription.

SUMMARY OF THE INVENTION

We describe here the expression of pygopus mRNA and Pygopus protein inestablished cancer cell lines and in patient tumors to assess its roleas a diagnostic and prognostic indicator, as well as disruptingstrategies that specifically target the activity of pygopus to determineits role in cancer.

Rational drug design utilizes emerging technologies that target cellularprocesses uniquely adopted by cancer cells for their survival. Study ofthe function of genes required for early embryonic development, forinstance, is a focal point in biomedical research becausedevelopmentally important genes also have a role in cancer.Understandably, controlled embryonic cellular activities such asproliferation and invasion/migration are activities necessary for cancercell survival. Conversely, many cancers result because of the abnormalactivation of cellular processes and functions that were once used inthe embryo but are no longer necessary for, and indeed are detrimentalto adult cell function.

We have determined, using molecular analyses and antisense RNAtechniques, that human Pygopus is a nuclear protein required for cellproliferation of human malignant tumors. This functional data issupported by expression analyses using both nucleic acid probes andPygopus specific antibodies, which demonstrate that Pygopus isconsistently and highly overexpressed in highly malignant cancer cellsincluding those of ovarian, breast, colon and uterine cervix. Unlike anyother protein, Pygopus is a novel, generalized marker and factorrequired for cancer cell survival. Therefore, non-expressing embryoniccells and non-expressing adult cells are insensitive to attempts whichdeplete Pygopus' function. As such, Pygopus can be used for diagnosticpurposes and as a specific pharmacological target to fight a wide rangeof cancers.

The Pygopus protein has two distinctive domains, a 50 amino acid stretchwithin the N-terminus referred to as the N-terminal homology domain(NHD) or N box and the C-terminal plant homeodomain (PHD). PHD motifs(also known as leukemia associated protein, or LAP, domains) are zincfinger-like domains of the concensus Cys4-His-Cys3 that are presentwithin a number of chromatin remodeling-type transcriptional regulators(FIG. 1).

The NHD of pygopus is the domain that activates Wnt signalling. Thisdomain comprises the N box (about 47 residues), and may extend to theN-terminal half of pygopus. For example, the NHD of human pygopus maycomprise up to about amino acids 1-232. The NHD domain may be usedwithout the endogenous nuclear localization signal (NLS), or theendogenous NLS may be replaced with a heterologous NLS. Thus in oneembodiment, the NHD comprises at least about 47 amino acids in theN-terminal half of pygopus, and retains the ability to activate Wntsignalling.

Pygopus and Pygopus-derived peptide and nucleic acid sequences may beused as a therapeutic (eg chemo, hormonal, etc.) target for cancer.Antibodies and other molecular devices related to Pygopus may be usedfor detection and diagnosis of all pre-tumor and tumor cells forprognostic information. Antisense RNA, RNAi and antibodies may be usedto deplete Pygopus function. Pygopus and Pygopus-derived peptide andnucleic acid sequences may be used to screen for cancer and precancercells.

Our highly sensitive antibodies and nucleic acid probes will be used tospecifically detect and differentiate cancer cells in, for instance, apathological specimen, a surgical biopsy, or other cytological-baseddiagnostic procedures such as pap smears. Thus new diagnostic reagentsfor clinical prognostic diagnosis of cancer are provided. Suchdiagnostic tools may be used to accurately assess tumor grade and stageas a prognostic indicator to more effectively and economically managecancer and pre-cancer patients.

We demonstrate that depletion of Pygopus specifically stops cancer cellgrowth. Thus molecules that specifically interact with and inactivatethe function of Pygopus would be useful for treating cancer.

We describe a method of screening for inhibitors of pygopus activity bytesting for the ability of a candidate inhibitor to bind specifically toNHD. Binding of the candidate to NHD would indicate that the candidateis a potential inhibitor of pygopus activity. That the candidate is apygopus inhibitor may be confirmed by testing for the candidate'sability to block transcriptional activation of Wnt-responsive genes,such as Cyclin D1, by NHD. One way of testing for transcriptionalactivation of Wnt-responsive genes is by using the TOPFLASH system (seefor example, Korinek et al 1997 Science 275:1784-1787).

We also describe antisense sequences against human pygopus, includinghPygo1 (SEQ ID NOs:3 and 4) and hPygo2 (SEQ ID NOs:1 and 2). Theantisense sequences may be those sequences specific to pygopus-2; i.e.antisense sequences that would bind to pygopus-2 and not to other humansequences. The antisense sequences include those which bind to thecoding region of human pygopus-2, or to the non-coding regions, inparticular the 3′ non-coding region. The antisense sequences may be atleast 10, 12, 15, 18, 20, 25, 30, 35 or 50 nucleotides long. It is notedthat hPygo1 is probably functionally interchangeable with hPygo2(Thompson, B. et al. Nat. Cell Biol. 4, 367-373 (2002)).

We also described proteins, other than full-length human pygopus, whichcomprise fragments of human pygopus. These fragments are useful at leastas antigens to elicit an immune response and produce antibodies. Thefragments include regions that are unique to human pygopus-2. Thefragments may be derived from amino acids 1-45, or 74-312 of humanpygopus-2. The fragments should be of sufficient size as to form afunctional epitope to elicit an antibody response. An epitope may be asshort as 8 to 10 amino acids.

The proteins comprising the fragments may be a fusion protein whichincludes a heterologous protein fused in frame to the fragment describedabove. The fragments, particularly short peptides of 8 to 30 aminoacids, may also be crosslinked to carrier molecules for eliciting anantibody response.

We also describe antibodies, both polyclonal and monoclonal, to humanpygopus. In one embodiment, the antibody binds to a fragment of humanpygopus-2. Such a fragment include regions of pygopus that are unique tohuman pygopus-2 such as those derived from amino acids 1-45, or 74-312of human pygopus-2. In a preferred embodiment, the antibody is ofsufficient titre as to be able to bind specifically to tumor cells insitu or in vivo.

We also describe methods of making antibodies to human pygopus. Themethod comprises eliciting an antibody response to a fragment of humanpygopus, as described above, or a fusion protein comprising a fragmentof human pygopus or a fragment of human pygopus linked to a carriermolecule.

We also describe a method for determining whether a cell is a cancercell. The method comprises assaying the cell for overexpression ofpygopus. In one embodiment, the cell is a human cell. In anotherembodiment, the cancer is ovarian, ovarian epithelial, breast, uterinecervical, cervical, lung, or colon cancer. The cell may be assayed forpygopus mRNA overexpression, or pygopus protein overexpression.

We also describe methods and kits for diagnosis of cancer. The methodand kits test for for overexpression of pygopus. In one embodiment, thecell is a human cell. In another embodiment, the cancer is ovarian,ovarian epithelial, breast, uterine cervical, cervical, lung, or coloncancer. The cell may be assayed for pygopus mRNA overexpression, orpygopus protein overexpression.

We also describe methods for attenuating growth of cancer cells thatexpress pygopus. The method comprises depleting the cell of pygopusactivity. In one embodiment, pygopus activity is depleted by means ofantisense polynucleotides. In another embodiment, pygopus activity isdepleted using RNA interference.

The present invention thus relates to a method for determining thepresence or absence of a cancer in a patient, the method comprising thesteps of: (a) determining the level of Pygopus gene expression in abiological sample obtained from a patient, and (b) comparing the levelof Pygopus gene expression in the biological sample to a predeterminedcut-off value, to determine whether Pygopus expression is higher in thebiological sample; therefrom determining the presence or absence ofcancer in the patient.

The present invention further relates to a method for monitoring theprogression of a cancer in a patient, the method comprising the stepsof: (a) determining the level of Pygopus gene expression in a biologicalsample obtained from a patient, and (b) comparing the level of Pygopusgene expression in the biological sample to a predetermined cut-offvalue, to determine whether Pygopus expression is higher in thebiological sample; and therefrom determining the presence or absence ofcancer in the patient; (c) repeating steps (a) and (b) using abiological sample obtained from the patient at a subsequent time; and(d) comparing the level of Pygopus gene expression detected in step (c)to the level of Pygopus gene expression detected in step (b); andtherefrom monitoring the progression of the cancer in the patient. Thepredetermined cut-off value may be the level of Pygopus gene expressionin a normal biological sample.

In certain embodiments, the cancer is ovarian cancer, and the biologicalsample is a tissue biopsy containing epithelial ovarian cells; or thecancer is breast cancer, and the biological sample is a tissue biopsycontaining mammary cells.

In certain embodiments, the Pygopus gene is hPygo2 as shown in SEQ IDNO:1, or hPygo1 as shown in SEQ ID NO:3.

In certain embodiments, the level of Pygopus gene expression isdetermined by the amount of Pygopus protein or by the amount of PygopusmRNA.

The present invention further relates to a kit for determining thepresence or absence of a cancer in a patient, the kit comprising areagent capable of detecting Pygopus protein or mRNA in a biologicalsample obtained from the patient, and instructions for using the reagentto determine whether the level of Pygopus gene expression in thebiological sample is higher compared to a predetermined cut-off value,and therefrom determining the presence or absence of cancer in thepatient.

In certain embodiments, the reagent is an antibody specifically reactiveto Pygopus protein.

In certain embodiments, the reagent is a polynucleotide capable ofbinding to a Pygopus gene or to a part of a Pygopus gene.

In certain embodiments, the predetermined cut-off value is the level ofPygopus gene expression in a normal biological sample.

The present invention further relates to human Pygopus polypeptide whichlacks the plant homeodomain (PHD) sequence and the N-terminal homologydomain (NHD) sequence.

In certain embodiments, the polypeptide is hPygo-2 (SEQ ID NO:2) lackingamino acids 89-328 or hPygo-1 (SEQ ID NO:4) lacking amino acids 85-341.

The present invention further relates to a nucleic acid encoding thepolypeptide of the invention, for example comprising nucleotides437-1156 of SEQ ID NO:1 or nucleotides 253-1023 of SEQ ID NO:3.

The present invention further relates to an antibody specificallyreactive with the polypeptide of the invention. The antibody may be amonoclonal antibody.

The present invention further relates to a method for obtaining acompound which inhibits tumor cell proliferation, wherein the tumor cellexpresses Pygopus, the method comprising: (a) testing a candidatecompound and selecting the compound for binding to an expressed productof a Pygopus gene; (b) testing the compound selected in (a) for itsability to inhibit Pygopus-mediated transcription activation of aWnt-responsive gene; and optionally (c) testing the compound selected in(b) in epithelial ovarian carcinoma or breast cancer cells for itsability to inhibit proliferation of the cells.

In certain embodiments, in step (a), the candidate compound is testedand selected for binding to a Pygopus protein.

In certain embodiments, in step (a), the candidate compound is testedand selected for binding to a Pygopus mRNA.

In certain embodiments, in step (b), the candidate compound is testedfor its ability to inhibit Pygopus-mediated transcription activation ofCyclin D1.

The present invention further relates to a method for obtaining anantisense polynucleotide which inhibits tumor cell proliferation,wherein the tumor cell express Pygopus, the method comprising: (a)providing a polynucleotide which is antisense to a Pygopus gene, orantisense to a portion of a Pygopus gene; (b) delivering thepolynucleotide into epithelial ovarian carcinoma or breast cancer cells;and (c) determining whether the delivered polynucleotide inhibitsproliferation of the cancer cells.

The present invention further relates to a method for obtaining acompound which inhibits tumor cell proliferation, wherein the tumor cellexpress Pygopus, the method comprising: (a) providing a shortinterfering RNA (siRNA) or siRNA-like molecule targeted to a Pygopusgene or to a portion of a Pygopus gene; (b) delivering the siRNA orsiRNA-like molecule into epithelial ovarian carcinoma or breast cancercells; and (c) determining whether the delivered siRNA or siRNA-likemolecule inhibits proliferation of the cancer cells.

The present invention further relates to a method for inhibiting tumorcell proliferation, the method comprising contacting the tumor cell witha proliferation-inhibiting amount of a compound which reduces Pygopusactivity in the cell.

In certain embodiments, the tumor cell is an epithelial ovariancarcinoma cell or breast cancer cell.

In certain embodiments, the compound reduces the ability of Pygopus toinhibit transcription activation of a Wnt-responsive gene.

In certain embodiments, the Wnt-responsive gene is Cyclin D1.

The present invention further relates to a method for inhibiting tumorcell proliferation, the method comprising delivering to the tumor cell aproliferation-inhibiting amount of a compound which reduces expressionof a Pygopus-encoding nucleic acid.

In certain embodiments, the compound is a polynucleotide which isantisense to a Pygopus gene, or antisense to a portion of a Pygopusgene.

In certain embodiments, the compound is a short interfering RNA (siRNA)or siRNA-like molecule targeted to a Pygopus gene or to a portion of aPygopus gene.

The present invention further relates to an antisense oligonucleotidetargeted to hPygo2 (SEQ ID NO:1) in the region from nucleotide 437 to1156 of SEQ ID NO:1, wherein said antisense oligonucleotide specificallyhybridizes with said region and reduces the expression of hPygo2.

The present invention further relates to an antisense oligonucleotidetargeted to hPygo1 (SEQ ID NO:3) in the region from nucleotide 253 to1023 of SEQ ID NO:3, wherein said antisense oligonucleotide specificallyhybridizes with said region and reduces the expression of hPygo1.

The present invention further relates to a short interfering RNA (siRNA)or siRNA-like molecule targeted to hPygo2 (SEQ ID NO:1) in the regionfrom nucleotide 437 to 1156 of SEQ ID NO:1, wherein said siRNA orsiRNA-like molecule reduces the expression of hPygo2.

The present invention further relates to a short interfering RNA (siRNA)or siRNA-like molecule targeted to hPygo1 (SEQ ID NO:3) in the regionfrom nucleotide 253 to 1023 of SEQ ID NO:3, wherein said siRNA orsiRNA-like molecule reduces the expression of hPygo1.

In certain embodiments, the antisense oligonucleotide has the sequenceselected from the group consisting of SEQ ID NOS:5-14.

In certain embodiments, the siRNA or siRNA-like molecule has thesequence selected from the group consisting of SEQ ID NOS:15-19.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Amino acid sequence of human hPygo2. The putative nuclearlocalisation signal (KKRRK) is in bold. The C-terminal PHD id doubleunderlined and the NHD is single underlined.

FIG. 2: Sequence alignment of human hPygo1 and hPygo2.

FIG. 3: Ability of anti-hPygo2 immune serum to recognize differentregions of hPygo2.

(a) Gal-4-hPygo2 fusion protein constructs.

(b) Western analysis of Gal-4-hPygo2 constructs transiently transfectedinto HeLa cells using anti-hPygo2 antiserum.

(c) Western analysis of Gal-4-hPygo2 constructs using an anti-Gal-4antibody.

(d) and (e) Similar experiments conducted with fusions to Flag peptide.

(f) Immunocytochemical analyses of four OvCa cell lines usinganti-hPygo2 antibodies and beta-catenin.

(g) Antisense oligonucleotides mapped to the full hPygo2 cDNA sequence.(h) Antisense oligonucleotides transfected into HeLa cells and RT-PCRanalysis performed to assess the relative knockdown of hPygo2 RNAlevels. Densitometric scan shows the relative hPygo2 levels compared tothe relative GAPDH levels. RT−, negative control, without reversetranscriptase.

FIG. 4: Consistent overexpression of hPygo2 in epithelial ovariancancer.

(a) Western analysis of normal (hOSE-1, -2) and malignant cell linesindicates that expression of Wnt signaling components is variable acrossdifferent cell lines.

(b) expression of hPygo2 RNA on northern blots (NB) and protein onimmunoblots (IB) indicates that they are highly overexpressed inmalignant EOC cell lines. In vitro synthesized hPygo2 (IVT) was used asa positive control.

(c) hPygo2 protein expression in tumors was graded according tointensity and frequency of tumor cell nuclei stained with hPygo2antiserum as compared to underlying stromal cells. Non-malignant ovarianepithelial adenomas are negative (−) for hPygo2. Staining of malignanttumors ranged from weak (+) to moderate (++) to strong (+++).

(d) Examples of hPygo2 and β-catenin expression in adjacent tumorsections. Upper pair shows strong nuclear staining of hPygo2 coincidentwith weak nuclear and moderate cytoplasmic β-catenin staining. Lowerpair indicates coincident moderate hygo2 nuclear staining and negativeβ-catenin staining.

FIG. 5: Demonstration of knockdown of protein in SK-OV-3 and OV-CAR-3EOC cell lines.

(a) Cells were transfected with either antisense hPygo2 (αs) ormismatched control (mm) oligonucleotides and compared to untransfected(Cont.) or mock transfected (Reag.) cells. Expression of either hPygo1or β-catenin was unaffected indicating specificity of the ON. GAPDHexpression was used as a loading control for RT-PCR and ERK-1 forimmunoblots.

(b) Both SK-OV-3 and OV-CAR-3 cells were transfected withβ-catenin-specific siRNA and two siRNAs (Hpy2A, D) for hPygo2.Expression of hPygo2 and β-catenin was assayed by immunoblot, usingERK-1 as a loading control and in vitro synthesized hPygo2 (IVT) wasused as a positive.

FIG. 6: Visualization of hPygo2 and β-catenin knockdown in EOC celllines by confocal microscopy. In the colour version of this Figure, redfluorescence indicates expression of b-catenin and green fluorescenceindicates hPygo2 expression.

Control transfected SK-OV-3 cells shows b-catenin associated primarilywith the plasma membrane while hPygo2 is exclusively in the nucleus.

(b) Cells stained with pre-immune hPygo2 serum.

(c) SK-OV-3 cells transfected with mismatched control ON.

(d) SKOV-3 cells transfected with antisense hPygo20N.

(e) Control OV-CAR-3 cells with plasma membrane-associated β-catenin andnuclear hPygo2.

(f) & (j) OV-CAR-3 cells stained with pre-immune serum.

(g) OV-CAR-3 cells transfected with mismatched ON.

(h) OV-CAR-3 cells transfected with with antisense hPygo2 ON.

(i) Non-specific siRNA does not affect β-catenin or hPygo2 expression inOV-CAR-3 cells.

(k) Knockdown of β-catenin in OV-CAR-3 cells does not affect hPygo2expression.

(1) Knockdown of hPygo2 in OV-CAR-3 cells. Arrowhead indicates a singlecell with normal hPygo2 expression.

FIG. 7: hPygo2 is required for EOC cell survival

(a) Growth assays of SK-OV-3 and OV-CAR-3 cells transfected withantisense hPygo2 (αs) and mismatched (mm) control ONs, 48 and 72 hoursafter transfection.

(b) DNA content of SK-OV-3 cells transfected with β-catenin and hPygo2siRNA compared to control and mock transfected (reagents) cellsindicates a higher proportion of sub-G1 cells in hPygo2-depleted cells,as measured by the areas under the curves.

FIG. 8: Immunohistochemical analysis of hPygo2 in breast tumors.

(a) Normal breast tissue negatively stained for hPygo2.

(b-d) Infiltrating ductal carcinomas stained with hPygo2. Weakcytoplasmic hPygo2 staining (b), strong cytoplasmic hPygo2 staining (c),Strong nuclear and moderate cytoplasmic hPygo2 staining. Scale=100micrometers.

FIG. 9: Expression of hPygo2, β-Catenin and Bcl-9 in cell lines. Levelsof RNA and protein were standardized using GAPDH and β-Actin.

(a) Expression of hPygo2 mRNA by northern analysis of total RNA. Thepositions of the 28s and 18s ribosomal RNAs are indicated.

(b) Immunoblot showing specificity of antibody for the hPygo2 protein.The approximate size of hPygo2 protein is 50 KDa, as indicated bymolecular weight markers are shown on the left. In vitro transcribed andtranslated full length hPygo2 protein (hPygo2) was used as a positivecontrol.

(c) Expression of hPygo2 and β-Catenin by western blot analysis of totalcell lysate of the various cell lines used.

(d) Expression of Pygo binding partner Bcl-9. Total RNA was analyzed byRT-PCR using primers specific to Bcl-9. −RT, negative control, withoutreverse transcriptase.

(d) Human Pygopus protein is expressed in malignant cancer cell linesindependently of Wnt-signalling factors. Total protein was extractedfrom eight different cell lines representing four different tumor typesand processed for Western analysis using antibodies against Wnt targetproteins and transducers, as well as anti-hPygo2 antibodies.

(f) Anti-hPygo2 antibodies were used for immuno-histochemical analysisof MCF-7 breast cancer cells and SK-OV-3 ovarian cancer cells.Anti-hPygo2 antibodies stain the nuclei specifically.

FIG. 10: Subcellular localization of hPygo2 and β-Catenin in normalbreast (Hs-574) and malignant breast cancer (Bt-474, Mcf-7) cells usingimmunofluorescence and confocal microscopy. Preimmune serum used at thesame dilution as hPygo2 immune serum for a negative control.

FIG. 11: Knockdown of β-Catenin in Mcf-7 cells by RNAi. Reagent control(Oligofectamine) and non-specific siRNA controls are indicated.

(a) Western blot analysis showing knockdown of β-Catenin protein inMcf-7 cells treated with siRNA. Loaded protein was standardized byreprobing blots with β-Actin.

(b) Cell proliferation 72 hours after initial treatment of cells withβ-Catenin siRNA. Results indicated are based on three experimentsperformed in triplicate.

FIG. 12: Knockdown of endogenous hPygo2 mRNA and protein using antisenseON performed in HeLa cells. Reagent control (Oligofectamine), antisenseXenopus Pygopus2 (non-specific), and four base mismatch (mismatch)controls are indicated. Levels of cDNA and protein were standardizedwith GAPDH and b-Actin. Expreiments were performed in triplicate.

(a) RT-PCR analysis of both human Pygo family members that werepreviously treated with antisense ON, showing specific knockdown ofhPygo2. RT−, negative control, without reverse transcriptase.

(b) Western blot analysis showing knockdown of hPygo2 protein.

FIG. 13: Knockdown of hPygo2 in Mcf-7 cells using antisense ONs. Reagentcontrol (Oligofectamine), antisense Xenopus Pygopus2 (non-specific), andfour base mismatch (mismatch) controls are indicated.

(a) Confirmation of hPygo2 protein knockdown by western blot analysis ofMcf-7 total cell lysate that was treated with antisense ON.

(b) Cell proliferation 72 hours after initial treatment of cells withantisense ON. Results indicated are based on three experiments performedin triplicate.

FIG. 14: Knockdown of hPygo2 in Mcf-7 cells with siRNA. Reagent control(Oligofectamine), Non-specific control siRNA (NS), β-Catenin and hPygo2Aand hPygo2D siRNAs are indicated. Cell growth and knockdown of proteinwas assayed at 72 hrs after transfection. Results indicated are based onthree experiments performed in triplicate.

FIG. 15: Anti-hPygo2 antibodies are used in immuno-histochemicalanalysis to identify malignant tumor cells from ovarian epithelial,breast, and lung cancer. Staining of archived tumors, as determined by alicenced pathologist, using anti-hPygo2 antibodies, indicates thatPygopus is specifically overexpressed in a variety of ovarian epithelial(A,C) tumors and in malignant breast (G) and lung cancer (H). Negativestaining with pre-immune and secondary antibody alone demonstratesspecificity of the antibody.

FIG. 16: Knockdown of endogenous hPygo2 using antisense ON in HeLacervical cancer cells. Reagent control (Oligofectamine), antisenseXenopus Pygopus2 (non-specific), and four base mismatch (mismatch)controls are indicated.

(a) HeLa cell numbers 48 and 72 hours after transfection with antisenseON.

(b) RT-PCR analysis of hPygo2 mRNA and of the related Pygo familymember, hPygo1. RT−, negative control, without reverse transcriptase.

(c) Western blot analysis of endogenous hPygo2 protein. Levels of cDNAand protein were standardized using GAPDH and β-Actin.

DETAILED DESCRIPTION OF EMBODIMENTS

(I) Polypeptides, Nucleic Acids and Uses:

The term “Pygopus” in the present context means nucleic acids andpolypeptides which are homologs of the gene identified as SEQ ID NO:1.In humans, there are at least two Pygopus genes, hPygo1 (SEQ ID NOS:3and 4) and hPygo2 (SEQ ID NOS:1 and 2); see FIGS. 1 and 2. Pygopus alsorefers to variants of the naturally occurring form of the gene, wherethe variants closely resemble the naturally occurring gene and retainthe function(s) of the naturally occurring gene.

The term “isolated polynucleotide or polypeptide” is defined as apolynucleotide or polypeptide removed from the environment in which itnaturally occurs. For example, a naturally-occurring DNA moleculepresent in the genome of a living bacteria or as part of a gene bank isnot isolated, but the same molecule separated from the remaining part ofthe bacterial genome, as a result of, e.g., a cloning event(amplification), is isolated. Typically, an isolated DNA molecule isfree from DNA regions (e.g., coding regions) with which it isimmediately contiguous at the 5′ or 3′ end, in the naturally occurringgenome. Such isolated polynucleotides may be part of a vector or acomposition and still be defined as isolated in that such a vector orcomposition is not part of the natural environment of suchpolynucleotide.

The polynucleotide of the invention is either RNA or DNA (cDNA, genomicDNA, or synthetic DNA), or modifications, variants, homologs orfragments thereof. The DNA is either double-stranded or single-stranded,and, if single-stranded, is either the coding strand or the non-coding(anti-sense) strand. By “polypeptide” or “protein” is meant any chain ofamino acids, regardless of length or post-translational modification(e.g., glycosylation or phosphorylation). Both terms are usedinterchangeably in the present application.

As used herein, “homologous amino acid sequence” is any polypeptidewhich is encoded, in whole or in part, by a nucleic acid sequence whichhybridizes at 25-35° C. below critical melting temperature (Tm), to anyportion of the nucleic acid sequence of SEQ ID No: 1 or 3. A homologousamino acid sequence is one that differs from an amino acid sequenceshown in SEQ ID No: 2 or 4 by one or more conservative amino acidsubstitutions. Such a sequence encompasses those which retain inherentcharacteristics of the polypeptide such as immunogenicity. Preferably,such a sequence is at least 75%, more preferably 80%, and mostpreferably 90% identical to SEQ ID No: 2 or 4.

Homologous amino acid sequences include sequences that are identical orsubstantially identical to SEQ ID No:2 or 4. By “amino acid sequencesubstantially identical” is meant a sequence that is at least 90%,preferably 95%, more preferably 97%, and most preferably 99% identicalto an amino acid sequence of reference and that preferably differs fromthe sequence of reference by a majority of conservative amino acidsubstitutions, i.e. substitutions among amino acids of the same class.

Homology is measured using sequence analysis software such as SequenceAnalysis Software Package of the Genetics Computer Group, University ofWisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis.53705. Amino acid sequences are aligned to maximize identity. Gaps maybe artificially introduced into the sequence to attain proper alignment.

In the present context, stringent conditions are achieved for bothpre-hybridizing and hybridizing incubations (i) within 4-16 hours at 42°C., in 6×SSC containing 50% formamide, or (ii) within 4-16 hours at 65°C. in an aqueous 6×SSC solution (1 M NaCl, 0.1 M sodium citrate (pH7.0)). Typically, hybridization experiments are performed at atemperature from 60 to 68° C., e.g. 65° C. At such a temperature,stringent hybridization conditions can be achieved in 6×SSC, preferablyin 2×SSC or 1×SSC, more preferably in 0.5×SSc, 0.3×SSC or 0.1×SSC (inthe absence of formamide). 1×SSC contains 0.15 M NaCl and 0.015 M sodiumcitrate.

Partial sequences of SEQ ID No: 2 or 4 or their homologous amino acidsequences are inherent to the full-length sequences. Such polypeptidefragments preferably are at least 12 amino acids in length, preferablyat least 15, 20, 25, 30, 35, 40, 45, 50 amino acids, more preferably atleast 55, 60, 65, 70, 75 amino acids, and most preferably at least 80,85, 90, 95, 100 amino acids in length.

Polynucleotides encoding polypeptide fragments and polypeptides havinglarge internal deletions are constructed using standard methods (Ausubelet al., Current Protocols in Molecular Biology, John Wiley & Sons Inc.,1994). Such methods include standard PCR, inverse PCR, restrictionenzyme treatment of cloned DNA molecules, or the method of Kunkel et al.(Kunkel et al. Proc. Natl. Acad. Sci. USA (1985) 82:448). Components forthese methods and instructions for their use are readily available fromvarious commercial sources.

In the present context, a fusion polypeptide is one that contains apolypeptide or a polypeptide derivative of the invention fused at the N-or C-terminal end to any other polypeptide (hereinafter referred to as apeptide tail). A simple way to obtain such a fusion polypeptide is bytranslation of an in-frame fusion of the polynucleotide sequences, i.e.,a hybrid gene. The hybrid gene encoding the fusion polypeptide isinserted into an expression vector which is used to transform ortransfect a host cell. Alternatively, the polynucleotide sequenceencoding the polypeptide or polypeptide derivative is inserted into anexpression vector in which the polynucleotide encoding the peptide tailis already present. Such vectors and instructions for their use arecommercially available.

The nucleic acid molecules encoding Pygopus and variants and fragmentsthereof, are useful for probes, primers, chemical intermediates, and inbiological assays. The nucleic acid molecules are useful as ahybridization probe for messenger RNA, transcript/cDNA and genomic DNAand to isolate cDNA and genomic clones that correspond to variants(alleles, orthologs, etc.) producing the same or related peptides.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are useful for expressing antigenic portionsof the proteins, as well as for designing antisense polynucleotides,siRNA-like molecules, or ribozymes corresponding to all, or a part, ofthe mRNA produced from the nucleic acid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of Pygopus, and are also useful for constructinghost cells expressing a part, or all, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided herein indicates that Pygopusis over-expressed in various human tumours. Accordingly, the probes canbe used to detect the presence of, or to determine levels of, Pygopus incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inPygopus expression relative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express Pygopus, such as by measuring a level ofnucleic acid encoding Pygopus in a sample of cells from a subject e.g.,mRNA or genomic DNA, or determining if the Pygopus gene has beenmutated.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate Pygopus gene expression. The invention thusprovides a method for identifying a compound that can be used to treat adisorder associated with expression of the Pygopus gene, particularlybiological and pathological processes that are mediated by Pygopus incells and tissues that express it. The method typically includesassaying the ability of the compound to modulate the expression of thePygopus gene and thus identify a compound that can be used to treat adisorder characterized by undesired Pygopus gene expression. The assayscan be performed in cell-based and cell-free systems. Cell-based assaysinclude cells naturally expressing the Pygopus gene or recombinant cellsgenetically engineered to express specific Pygopus sequences.

The assay for Pygopus gene expression can involve direct assay ofnucleic acid levels, such as mRNA levels, or on collateral compoundsinvolved in the Wnt-signalling pathway. Further, the expression of genesthat are up- or down-regulated in response to Wnt signalling pathway canalso be assayed. In this embodiment the regulatory regions of thesegenes can be operably linked to a reporter gene such as luciferase.

Thus, modulators of Pygopus gene expression can be identified in amethod wherein a cell is contacted with a test compound and theexpression of mRNA determined. The level of expression of Pygopus mRNAin the presence of the test compound is compared to the level ofexpression of Pygopus mRNA in the absence of the test compound. The testcompound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the test compound than in its absence, the test compound isidentified as a stimulator of nucleic acid expression. When nucleic acidexpression is statistically significantly less in the presence of thetest compound than in its absence, the test compound is identified as aninhibitor of nucleic acid expression.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in Pygopus gene expression, and particularly inqualitative changes that lead to cancer pathology. The nucleic acidmolecules can be used to detect mutations in Pygopus genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the Pygopus gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the Pygopus gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of Pygopus.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantPygopus gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242 (1985));Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al, PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The invention also encompasses kits for detecting the presence of akinase nucleic acid in a biological sample. The kit can comprisereagents such as a labeled or labelable nucleic acid or agent capable ofdetecting Pygopus-2 gene or mRNA in a biological sample; means fordetermining the amount of Pygopus mRNA in the sample; and means forcomparing the amount of Pygopus mRNA in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect Pygopus mRNAor DNA.

II. Antisense Polynucleotides

In alternative embodiments, the invention provides antisense moleculesand ribozymes for exogenous administration to effect the degradationand/or inhibition of the translation of Pygopus mRNA. Examples oftherapeutic antisense oligonucleotide applications, incorporated hereinby reference, include: U.S. Pat. No. 5,135,917, issued Aug. 4, 1992;U.S. Pat. No. 5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617,issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24, 1992; U.S.Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat. No. 5,194,428, issuedMar. 16, 1993; U.S. Pat. No. 4,806,463, issued Feb. 21, 1989; U.S. Pat.No. 5,286,717 issued Feb. 15, 1994; U.S. Pat. No. 5,276,019 and U.S.Pat. No. 5,264,423.

Preferably, in antisense molecules, there is a sufficient degree ofcomplementarity to the Pygopus mRNA to avoid non-specific binding of theantisense molecule to non-target sequences under conditions in whichspecific binding is desired, such as under physiological conditions inthe case of in vivo assays or therapeutic treatment or, in the case ofin vitro assays, under conditions in which the assays are conducted. Thetarget mRNA for antisense binding may include not only the informationto encode a protein, but also associated ribonucleotides, which forexample form the 5′-untranslated region, the 3′-untranslated region, the5′ cap region and intron/exon junction ribonucleotides. A method ofscreening for antisense and ribozyme nucleic acids that may be used toprovide such molecules is disclosed in U.S. Pat. No. 5,932,435.

As used herein, the term “target nucleic acid” encompass DNA encodingRNA (including pre-mRNA and mRNA) transcribed from such DNA, and alsocDNA derived from such RNA. The specific hybridization of an oligomericcompound with its target nucleic acid interferes with the normalfunction of the nucleic acid. This modulation of function of a targetnucleic acid by compounds which specifically hybridize to it isgenerally referred to as “antisense”. The functions of DNA to beinterfered with include replication and transcription. The functions ofRNA to be interfered with include all vital functions such as, forexample, translocation of the RNA to the site of protein translation,translocation of the RNA to sites within the cell which are distant fromthe site of RNA synthesis, translation of protein from the RNA, splicingof the RNA to yield one or more mRNA species, and catalytic activitywhich may be engaged in or facilitated by the RNA. The overall effect ofsuch interference with target nucleic acid function is modulation of theexpression of Pygopus. In the context of the present invention,“modulation” means either an increase (stimulation) or a decrease(inhibition) in the expression of a gene. In the context of the presentinvention, inhibition is the preferred form of modulation of geneexpression and mRNA is a preferred target.

“Targeting” an antisense compound to a particular nucleic acid, in thecontext of this invention, is a multistep process. The process usuallybegins with the identification of a nucleic acid sequence whose functionis to be modulated. In the present invention, the target is a nucleicacid molecule encoding Pygopus. The targeting process also includesdetermination of a site or sites within this gene for the antisenseinteraction to occur such that the desired effect, e.g., detection ormodulation of expression of the protein, will result. Within the contextof the present invention, a preferred intragenic site is the regionencompassing the translation initiation or termination codon of the openreading frame (ORF) of the gene. Since, as is known in the art, thetranslation initiation codon is typically 5′-AUG (in transcribed mRNAmolecules; 5′-ATG in the corresponding DNA molecule), the translationinitiation codon is also referred to as the “AUG codon,” the “startcodon” or the “AUG start codon”. A minority of genes have a translationinitiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, theterms “translation initiation codon” and “start codon” can encompassmany codon sequences, even though the initiator amino acid in eachinstance is typically methionine (in eukaryotes) or formylmethionine (inprokaryotes). It is also known in the art that eukaryotic andprokaryotic genes may have two or more alternative start codons, any oneof which may be preferentially utilized for translation initiation in aparticular cell type or tissue, or under a particular set of conditions.In the context of the invention, “start codon” and “translationinitiation codon” refer to the codon or codons that are used in vivo toinitiate translation of an mRNA molecule transcribed from a geneencoding Pygopus, regardless of the sequence(s) of such codons.

It is also known in the art that a translation termination codon (or“stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA,5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAGand 5′-TGA, respectively). The terms “start codon region” and“translation initiation codon region” refer to a portion of such an mRNAor gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationinitiation codon. Similarly, the terms “stop codon region” and“translation termination codon region” refer to a portion of such anmRNA or gene that encompasses from about 25 to about 50 contiguousnucleotides in either direction (i.e., 5′ or 3′) from a translationtermination codon.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Other target regions include the 5′ untranslatedregion (5′ UTR), known in the art to refer to the portion of an mRNA inthe 5′ direction from the translation initiation codon, and thusincluding nucleotides between the 5′ cap site and the translationinitiation codon of an mRNA or corresponding nucleotides on the gene,and the 3′ untranslated region (3′ UTR), known in the art to refer tothe portion of an mRNA in the 3′ direction from the translationtermination codon, and thus including nucleotides between thetranslation termination codon and 3′ end of an mRNA or correspondingnucleotides on the gene. The 5′ cap of an mRNA comprises anN7-methylated guanosine residue joined to the 5′-most residue of themRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA isconsidered to include the 5′ cap structure itself as well as the first50 nucleotides adjacent to the cap. The 5′ cap region may also be apreferred target region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. mRNA splice sites, i.e., intron-exonjunctions, may also be preferred target regions, and are particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular mRNA splice product isimplicated in disease. Aberrant fusion junctions due to rearrangementsor deletions are also preferred targets. mRNA transcripts produced viathe process of splicing of two (or more) mRNAs from different genesources are known as “fusion transcripts”. It has also been found thatintrons can be effective, and therefore preferred, target regions forantisense compounds targeted, for example, to DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “mRNA variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic andextronic regions.

Upon excision of one or more exon or intron regions or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites.

Once one or more target sites have been identified, oligonucleotides arechosen which are sufficiently complementary to the target, i.e.,hybridize sufficiently well and with sufficient specificity, to give thedesired effect.

In the context of this invention, “hybridization” means hydrogenbonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary nucleoside or nucleotide bases.For example, adenine and thymine are complementary nucleobases whichpair through the formation of hydrogen bonds. “Complementary,” as usedherein, refers to the capacity for precise pairing between twonucleotides. For example, if a nucleotide at a certain position of anoligonucleotide is capable of hydrogen bonding with a nucleotide at thesame position of a DNA or RNA molecule, then the oligonucleotide and theDNA or RNA are considered to be complementary to each other at thatposition. The oligonucleotide and the DNA or RNA are complementary toeach other when a sufficient number of corresponding positions in eachmolecule are occupied by nucleotides which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of complementarity orprecise pairing such that stable and specific binding occurs between theoligonucleotide and the DNA or RNA target.

An antisense compound is specifically hybridizable when binding of thecompound to the target DNA or RNA molecule interferes with the normalfunction of the target DNA or RNA to cause a loss of activity, and thereis a sufficient degree of complementarity to avoid non-specific bindingof the antisense compound to non-target sequences under conditions inwhich specific binding is desired, i.e., under physiological conditionsin the case of in vivo assays or therapeutic treatment, and in the caseof in vitro assays, under conditions in which the assays are performed.

Antisense and other compounds of the invention, which hybridize to thetarget and inhibit expression of the target, are identified throughexperimentation, and representative sequences of these compounds arehereinbelow identified as preferred embodiments of the invention. Thesites to which these preferred antisense compounds are complementary arehereinbelow referred to as “hybridization-accessible sites” and aretherefore preferred sites for targeting. As used herein the term“hybridization-accessible site” is defined as at least an 8-nucleobaseportion of a region of a gene that is accessible for hybridization witha complementary sequence of nucleic acid.

While the specific sequences of particular hybridization-accessiblesites can be represented by the reverse complement of the antisenseoligonucleotides set forth in Table 2, one of skill in the art willrecognize that these serve to illustrate and describe particularembodiments within the scope of the present invention. Additionalhybridization-accessible sites may be identified by one having ordinaryskill.

Stretches of at least eight (8) consecutive nucleobases selected fromwithin the illustrative hybridization-accessible sites are considered tobe suitable hybridization-accessible sites as well, albeit exhibitinglower Tm values. Also, stretches of DNA or RNA that are about 8 to about80 consecutive nucleobases and that comprise some portion of the 5′- or3′-terminal sequence of a hybridization-accessible site will also beconsidered hybridization-accessible site for purposes of this invention.Exemplary good hybridization-accessible sites include DNA or RNAsequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one a hybridization-accessible site (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately upstream of the 5′-terminus of the hybridization-accessiblesite and continuing until the DNA or RNA contains about 8 to about 80nucleobases). Similarly good hybridization-accessible sites arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of ahybridization-accessible site (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 3′-terminus of the hybridization-accessible site andcontinuing until the target site contains about 8 to about 80nucleobases).

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense oligonucleotideshave been employed as therapeutic moieties in the treatment of diseasestates in animals and man. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat oligonucleotides can be useful therapeutic modalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans.

In the context of this invention, the term “oligonucleotide” refers toan oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleicacid (DNA) or mimetics thereof. This term includes oligonucleotidescomposed of naturally-occurring nucleobases, sugars and covalentinternucleoside (backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases.

While antisense oligonucleotides are a preferred form of antisensecompound, the present invention comprehends other oligomeric antisensecompounds, including but not limited to oligonucleotide mimetics such asare described below. The antisense compounds in accordance with thisinvention preferably comprise from about 8 to about 80 nucleobases (i.e.from about 8 to about 80 linked nucleosides). Particularly preferredantisense compounds are antisense oligonucleotides from about 8 to about50 nucleobases, even more preferably those comprising from about 12 toabout 30 nucleobases. Antisense compounds include ribozymes, externalguide sequence (EGS) oligonucleotides (oligozymes), and other shortcatalytic RNAs or catalytic oligonucleotides which hybridize to thetarget nucleic acid and modulate its expression.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include DNA or RNA sequencesthat comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the same DNAor RNA beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the DNA or RNA contains about 8 toabout 80 nucleobases). Similarly preferred antisense compounds arerepresented by DNA or RNA sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same DNA or RNA beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the DNA or RNA contains about 8 to about 80 nucleobases).

Antisense and other compounds of the invention, which hybridize to thetarget and inhibit expression of the target, are identified throughexperimentation, and representative sequences of these compounds areherein identified as preferred embodiments of the invention. Whilespecific sequences of the antisense compounds are set forth herein, oneof skill in the art will recognize that these serve to illustrate anddescribe particular embodiments within the scope of the presentinvention.

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure,however, open linear structures are generally preferred. In addition,linear structures may also have internal nucleobase complementarity andmay therefore fold in a manner as to produce a double strandedstructure. Within the oligonucleotide structure, the phosphate groupsare commonly referred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 51 phosphodiester linkage.

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Antisense molecules (oligonucleotides) of the invention may includethose which contain intersugar backbone linkages such asphosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages, phosphorothioates and those with CH₂—NH—O—CH₂,CH₂—N(CH₃)—0—CH₂ (known as methylene (methylimino) or MMI backbone),CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and 0—N(CH₃)—CH₂—CH₂ backbones(where phosphodiester is 0—P—O—CH₂). Oligonucleotides having morpholinobackbone structures may also be used (U.S. Pat. No. 5,034,506). Inalternative embodiments, antisense oligonucleotides may have a peptidenucleic acid (PNA, sometimes referred to as “protein nucleic acid”)backbone, in which the phosphodiester backbone of the oligonucleotidemay be replaced with a polyamide backbone wherein nucleosidic bases arebound directly or indirectly to aza nitrogen atoms or methylene groupsin the polyamide backbone (Nielsen et al., 1991, Science 254:1497 andU.S. Pat. No. 5,539,082). The phosphodiester bonds may be substitutedwith structures which are chiral and enantiomerically specific. Personsof ordinary skill in the art will be able to select other linkages foruse in practice of the invention.

Preferred modified oligonucleotide backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphateshaving normal 3′-5′ linkages, 2′-5′ linked analogs of these, and thosehaving inverted polarity wherein one or more internucleotide linkages isa 31 to 31, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotideshaving inverted polarity comprise a single 3′ to 3′ linkage at the3′-most internucleotide linkage i.e. a single inverted nucleosideresidue which may be abasic (the nucleobase is missing or has a hydroxylgroup in place thereof). Various salts, mixed salts and free acid formsare also included.

Oligonucleotides may also include species which include at least onemodified nucleotide base. Thus, purines and pyrimidines other than thosenormally found in nature may be used. Similarly, modifications on thepentofuranosyl portion of the nucleotide subunits may also be effected.Examples of such modifications are 2′-O-alkyl- and2′-halogen-substituted nucleotides. Some specific examples ofmodifications at the 2′ position of sugar moieties which are useful inthe present invention are OH, SH, SCH₃, F, OCN, O(CH₂)_(n)NH₂ orO(CH₂)_(n) CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl,substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-,S-, or N-alkyl; O—, S—, or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃;NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;polyalkylamino; substituted silyl; an RNA cleaving group; a reportergroup; an intercalator; a group for improving the pharmacokineticproperties of an oligonucleotide; or a group for improving thepharmacodynamic properties of an oligonucleotide and other substituentshaving similar properties. One or more pentofuranosyl groups may bereplaced by another sugar, by a sugar mimic such as cyclobutyl or byanother moiety which takes the place of the sugar.

In some embodiments, the antisense oligonucleotides in accordance withthis invention may comprise from about 5 to about 100 nucleotide units.As will be appreciated, a nucleotide unit is a base-sugar combination(or a combination of analogous structures) suitably bound to an adjacentnucleotide unit through phosphodiester or other bonds forming a backbonestructure.

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Any other means for such synthesis known in theart may additionally or alternatively be employed. It is well known touse similar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

III. RNA Interference

Expression of a Pygopus-encoding nucleic acid or fragment thereof may beinhibited or prevented using RNA interference (RNAi) technology, a typeof post-transcriptional gene silencing. RNAi may be used to create apseudo “knockout” or “knock down”, i.e. a system in which the expressionof the product encoded by a gene or coding region of interest isreduced, resulting in an overall reduction of the activity of theencoded product in a system. As such, RNAi may be performed to target anucleic acid of interest or fragment or variant thereof, to in turnreduce its expression and the level of activity of the product which itencodes. Such a system may be used for functional studies of theproduct, as well as to treat disorders related to the activity of such aproduct. RNAi is described in for example Hammond et al. (2001), Sharp(2001), Caplen et al. (2001), Sedlak (2000) and published US patentapplications 20020173478 (Gewirtz; published Nov. 21, 2002) and20020132788 (Lewis et al.; published Nov. 7, 2002), all of which areherein incorporated by reference. Reagents and kits for performing RNAiare available commercially from for example Ambion Inc. (Austin, Tex.,USA) and New England Biolabs Inc. (Beverly, Mass., USA).

This invention relates to compounds, compositions, and methods usefulfor modulating expression of Pygopus by RNA interference (RNAi) usingshort interfering nucleic acid (siNA). A siNA of the invention can beunmodified or chemically modified. A siNA of the instant invention canbe chemically synthesized, expressed from a vector, or enzymaticallysynthesized. The use of chemically-modified siNA is expected to improvevarious properties of native siNA molecules through increased resistanceto nuclease degradation in vivo and/or improved cellular uptake. ThesiNA molecules of the instant invention provide useful reagents andmethods for a variety of therapeutic and diagnostic applications.

The invention features one or more siNA molecules and methods thatindependently or in combination modulate the expression of Pygopus.

The invention also features a siNA molecule comprising 2′-5′internucleotide linkages. The 21-51 internucleotide linkage(s) can be at3′-end the 5′-end, the 3″-end, or both of the 5′- and 3′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically modified, wherein each strand is between about 18 andabout 27 (e.g., about 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27)nucleotides in length, wherein the duplex has between about 18 and about23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs.

In another embodiment, a siNA molecule of the invention comprises asingle-stranded hairpin structure, wherein the siNA is between about 36and about 70 (e.g., about 36, 40, 45, 50, 55, 60, 65, or 70) nucleotidesin length having between about 18 and about 23 (e.g., about 18, 19, 20,21, 22, or 23) base pairs, and wherein the siNA can include a chemicalmodification to improve various properties of native siNA molecules.

In another embodiment, a linear hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. For example, a linear hairpin siNA moleculeof the invention is designed such that degradation of the loop portionof the siNA molecule in vivo can generate a double-stranded siNAmolecule with 3′-terminal overhangs, such as 3′-terminal nucleotideoverhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is between about 38 andabout 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides inlength having between about 18 and about 23 (e.g., about 18, 19, 20, 21,22, or 23) base pairs, and wherein the siNA can include a chemicalmodification to improve various properties of native siNA molecules.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of mediating RNA interference or gene silencing.For example the siNA can be a double-stranded polynucleotide moleculecomprising self-complementary sense and antisense regions, wherein theantisense region comprises complementarity to a target nucleic acidmolecule. The siNA can be a single-stranded hairpin polynucleotidehaving self-complementary sense and antisense regions, wherein theantisense region comprises complementarity to a target nucleic acidmolecule. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises complementarity to a target nucleic acid molecule, andwherein the circular polynucleotide can be processed either in vivo orin vitro to generate an active siNA capable of mediating RNAi. As usedherein, siNA molecules need not be limited to those molecules containingonly RNA, but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. In certain embodiments short interfering nucleic acids donot require the presence of nucleotides having a 2′-hydroxy group formediating RNAi and as such, short interfering nucleic acid molecules ofthe invention optionally do not contain any ribonucleotides (e.g.,nucleotides having a 2′-OH group). The modified short interferingnucleic acid molecules of the invention can also be referred to as shortinterfering modified oligonucleotides “siMON”. As used herein, the termsiNA is meant to be equivalent to other terms used to describe nucleicacid molecules that are capable of mediating sequence specific RNAi, forexample short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA, short hairpin RNA (shRNA), short interfering oligonucleotide,short interfering nucleic acid, short interfering modifiedoligonucleotide, chemically-modified siRNA, post-transcriptional genesilencing RNA (ptgsRNA), and others. In addition, as used herein, theterm RNAi is meant to be equivalent to other terms used to describesequence specific RNA interference, such as post-transcriptional genesilencing.

By “modulate” is meant that the expression of the gene, or level of RNAmolecule or equivalent RNA molecules encoding one or more proteins, oractivity of one or more proteins is up-regulated or down-regulated, suchthat expression, level, or activity is greater than or less than thatobserved in the absence of the modulator. For example, the term“modulate” can mean “inhibit” but the use of the word “modulate” is notlimited to this definition.

By “inhibit” is meant that the activity of a gene expression product orlevel of RNAs or equivalent RNAs encoding one or more gene products isreduced below that observed in the absence of the nucleic acid moleculeof the invention. In one embodiment, inhibition with a siNA moleculepreferably is below that level observed in the presence of an inactiveor attenuated molecule that is unable to mediate an RNAi response.

The initial agent for RNAi in some systems is thought to be dsRNAmolecule corresponding to a target nucleic acid. The dsRNA is thenthought to be cleaved into short interfering RNAs (siRNAs) which are21-23 nucleotides in length (19-21 bp duplexes, each with 2 nucleotide3′ overhangs). The enzyme thought to effect this first cleavage step hasbeen referred to as “Dicer” and is categorized as a member of the RNaseIII family of dsRNA-specific ribonucleases. Alternatively, RNAi may beeffected via directly introducing into the cell, or generating withinthe cell by introducing into the cell a suitable precursor (e.g. vectorencoding precursor(s), etc.) of such an siRNA or siRNA-like molecule. AnsiRNA may then associate with other intracellular components to form anRNA-induced silencing complex (RISC). The RISC thus formed maysubsequently target a transcript of interest via base-pairinginteractions between its siRNA component and the target transcript byvirtue of homology, resulting in the cleavage of the target transcriptapproximately 12 nucleotides from the 3′ end of the siRNA. Thus thetarget mRNA is cleaved and the level of protein product it encodes isreduced.

The siNA molecules of the invention can be designed to inhibit Pygopusgene expression through RNAi targeting of a variety of RNA molecules. Inone embodiment, the siNA molecules of the invention are used to targetvarious RNAs corresponding to a Pygopus gene. Non-limiting examples ofsuch RNAs include messenger RNA (mRNA), alternate RNA splice variants oftarget gene(s), post-transcriptionally modified RNA of target gene(s),pre-mRNA of target gene(s), and/or RNA templates. If alternate splicingproduces a family of transcripts that are distinguished by usage ofappropriate exons, gene expression can be inhibited through theappropriate exons to specifically inhibit or to distinguish among thefunctions of gene family members.

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a family of Pygopus genes.As such, siNA molecules targeting multiple Pygopus genes can provideincreased effect.

RNAi may be effected by the introduction of suitable in vitrosynthesized siRNA or siRNA-like molecules into cells. RNAi may forexample be performed using chemically-synthesized RNA. Alternatively,suitable expression vectors may be used to transcribe such RNA either invitro or in vivo. In vitro transcription of sense and antisense strands(encoded by sequences present on the same vector or on separate vectors)may be effected using for example T7 RNA polymerase, in which case thevector may comprise a suitable coding sequence operably-linked to a T7promoter. The in vitro-transcribed RNA may in embodiments be processed(e.g. using E. coli RNase III) in vitro to a size conducive to RNAi. Thesense and antisense transcripts are combined to form an RNA duplex whichis introduced into a target cell of interest. Other vectors may be used,which express small hairpin RNAs (shRNAs) which can be processed intosiRNA-like molecules. Various vector-based methods are described in theart. Various methods for introducing such vectors into cells, either invitro or in vivo (e.g. gene therapy) are known in the art.

Accordingly, in some embodiments Pygopus expression may be inhibited byintroducing into or generating within a cell an siRNA or siRNA-likemolecule corresponding to a Pygopus-encoding nucleic acid or fragmentthereof, or to an nucleic acid homologous thereto. “siRNA-like molecule”refers to a nucleic acid molecule similar to an siRNA (e.g. in size andstructure) and capable of eliciting siRNA activity, i.e. to effect theRNAi-mediated inhibition of expression. In various embodiments such amethod may entail the direct administration of the siRNA or siRNA-likemolecule into a cell, or use of the vector-based methods describedabove. In an embodiment, the siRNA or siRNA-like molecule is less thanabout 30 nucleotides in length. In a further embodiment, the siRNA orsiRNA-like molecule is about 21-23 nucleotides in length. In anembodiment, siRNA or siRNA-like molecule comprises a 19-21 bp duplexportion, each strand having a 2 nucleotide 3′ overhang. In embodiments,the siRNA or siRNA-like molecule is substantially identical to aPygopus-encoding nucleic acid or a fragment or variant (or a fragment ofa variant) thereof. Such a variant is capable of encoding a proteinhaving Pygopus-like activity. In embodiments, the sense strand of thesiRNA or siRNA-like molecule is substantially identical to the Pygopusnucleotide sequence, or a fragment thereof (RNA having U in place of Tresidues of the DNA sequence).

IV. Delivery of Nucleic Acid Molecules into Cells

A siNA molecule of the invention can be adapted for use to inhibitcancer cell proliferation, alone or in combination with other therapies.For example, a siNA molecule can comprise a delivery vehicle, includingliposomes, for administration to a subject, carriers and diluents andtheir salts, and/or can be present in pharmaceutically acceptableformulations.

Methods for the delivery of nucleic acid molecules are described inAkhtar et al., 1992, Trends Cell Bio., 2, 139; Delivery Strategies forAntisense Oligonucleotide Therapeutics, ed. Akhtar, 1995, Maurer et al.,1999, Mol. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb.Exp. Pharmacol., 137, 165-192; and Lee et al., 2000, ACS Symp. Ser.,752, 184-192, all of which are incorporated herein by reference.Beigelman et al., U.S. Pat. No. 6,395,713, and Sullivan et al., PCT WO94/02595, further describe the general methods for delivery of nucleicacid molecules. These protocols can be utilized for the delivery ofvirtually any nucleic acid molecule.

Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as hydrogels, cyclodextrins,biodegradable nanocapsules, and bioadhesive microspheres, or byproteinaceous vectors (O'Hare and Normand, International PCT PublicationNo. WO 00/53722). Alternatively, the nucleic acid/vehicle combination islocally delivered by direct injection or by use of an infusion pump.Direct injection of the nucleic acid molecules of the invention, whethersubcutaneous, intramuscular, or intradermal, can take place usingstandard needle and syringe methodologies, or by needle-freetechnologies such as those described in Conry et al., 1999, Clin. CancerRes., 5, 2330-2337 and Barry et al., International PCT Publication No.WO 99/31262. The molecules of the instant invention can be used aspharmaceutical agents. Pharmaceutical agents prevent, modulate theoccurrence, or treat (alleviate a symptom to some extent, preferably allof the symptoms) of a disease state in a patient.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedinto a patient by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as tablets, capsules orelixirs for oral administration, suppositories for rectaladministration, sterile solutions, suspensions for injectableadministration, and the other compositions known in the art.

The present invention also includes pharmaceutically acceptableFormulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonicacid.

By “pharmaceutically acceptable formulation” is meant, a composition orformulation that allows for the effective distribution of the nucleicacid molecules of the instant invention in the physical location mostsuitable for their desired activity.

The invention also features the use of the composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes).These formulations offer a method for increasing the accumulation ofdrugs in target tissues. This class of drug carriers resistsopsonization and elimination by the mononuclear phagocytic system (MPSor RES), thereby enabling longer blood circulation times and enhancedtissue exposure for the encapsulated drug. Such liposomes have beenshown to accumulate selectively in tumors, presumably by extravasationand capture in the neovascularized target tissues. The long-circulatingliposomes enhance the pharmacokinetics and pharmacodynamics of DNA andRNA, particularly compared to conventional cationic liposomes which areknown to accumulate in tissues of the MPS. Long-circulating liposomesare also likely to protect drugs from nuclease degradation to a greaterextent compared to cationic liposomes, based on their ability to avoidaccumulation in metabolically aggressive MPS tissues such as the liverand spleen.

V. Screening Assays

In another aspect, the invention relates to the use of Pygopus as atarget in screening assays that may be used to identify compounds thatare useful as inhibitors of Pygopus for the prevention or treatment ofcancer. In some embodiments, such an assay may comprise the steps of:

(a) providing a test compound;

(b) providing a source of Pygopus; and

(c) measuring Pygopus activity in the presence versus the absence of thetest compound, wherein a lower measured activity in the presence of thetest compound indicates that the compound is an inhibitor ofPygopus-dependent signal and may be useful for the prevention and/ortreatment of cancer.

“Pygopus activity” as used herein refers to any type of observedphenomenon which can be attributed to Pygopus, via for example thetranscriptional activation of Wnt-responsive genes or inhibition ofcancer cell proliferation. Transcriptional activation of Wnt-responsivegenes may be assayed using any methods known in the art. Usually suchmethods involve measuring the activity of a reporter gene operablylinked to a promoter, itself operably linked to Wnt-responsiveregulatory regions normally present in genes activated by the Wntpathway. In one embodiment, this transcriptional activity is measuredwith a luciferase reporter (TOPFLASH, provided by Upstate: CellSignalling Solutions, Charlottesville Va., USA; B-catenin Luciferasereporter construct; Korinek, V. et al., 1997, Science 275:1784-1787,incorporated herein by reference) that contains multiple T-cell Factor(TCF) binding sites, which are directly activated by the TCF/b-catenincomplex. To control for non-specific repression or activation, areporter with mutant Lef-1 binding sites (FOPFLASH) was substituted forTOPFLASH, and luciferase activity measured from the TOPFLASH reporter isnormalized accordingly.

The assay methods of the invention may be used to identify compoundscapable of modulating, e.g. inhibiting, Pygopus, in a biological system.In embodiments, the above noted biological system may be a mammal, suchas a human, or a suitable animal model system such as Xenopus.

The invention further provides a method of identifying a compound forthe prevention and/or treatment of cancer based on the identification ofa compound capable of modulating (e.g. inhibiting) Pygopus expression.Such a method may comprise assaying Pygopus gene expression in thepresence versus the absence of a test compound. Such gene expression maybe measured by detection of the corresponding RNA or protein, or via theuse of a suitable reporter construct comprising a transcriptionalregulatory element(s) normally associated with such a Pygopus gene,operably-linked to a reporter gene. A first nucleic acid sequence is“operably-linked” with a second nucleic acid sequence when the firstnucleic acid sequence is placed in a functional relationship with thesecond nucleic acid sequence. For instance, a promoter isoperably-linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequences. Generally,operably-linked DNA sequences are contiguous and, where necessary tojoin two protein coding regions, in reading frame. However, since forexample enhancers generally function when separated from the promotersby several kilobases and intronic sequences may be of variable lengths,some polynucleotide elements may be operably-linked but not contiguous.

“Transcriptional regulatory element” is a generic term that refers toDNA sequences, such as initiation and termination signals, enhancers,and promoters, splicing signals, polyadenylation signals which induce orcontrol transcription of protein coding sequences with which they areoperably-linked. The expression of such a reporter gene may be measuredon the transcriptional or translational level, e.g. by the amount of RNAor protein produced. RNA may be detected by for example Northernanalysis or by the reverse transcriptase-polymerase chain reaction(RT-PCR) method (see for example Sambrook et al (1989) MolecularCloning: A Laboratory Manual (second edition), Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA). Protein levels may bedetected either directly using affinity reagents (e.g. an antibody orfragment thereof [for methods, see for example Harlow, E. and Lane, D(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.]; a ligand which binds the protein) orby other properties (e.g. fluorescence in the case of green fluorescentprotein) or by measurement of the protein's activity, which may entailenzymatic activity to produce a detectable product (e.g. with alteredspectroscopic properties) or a detectable phenotype (e.g. alterations incell growth). Suitable reporter genes include but are not limited tochloramphenicol acetyltransferase, beta-D galactosidase, luciferase, orgreen fluorescent protein.

The above-noted methods and assays may be employed either with a singletest compound or a plurality or library (e.g. a combinatorial library)of test compounds. In the latter case, synergistic effects provided bycombinations of compounds may also be identified and characterized. Theabove-mentioned compounds may be used for inhibiting Pygopus, and forthe prevention and/or treatment of cancer, or may be used as leadcompounds for the development and testing of additional compounds havingimproved specificity, efficacy and/or pharmacological (e.g.pharmacokinetic) properties. In certain embodiments, one or a pluralityof the steps of the screening/testing methods of the invention may beautomated.

Such assay systems may comprise a variety of means to enable andoptimize useful assay conditions. Such means may include but are notlimited to: suitable buffer solutions, for example, for the control ofpH and ionic strength and to provide any necessary components foroptimal Pygopus activity and stability (e.g. protease inhibitors),temperature control means for optimal Pygopus activity and or stability,and detection means to enable the detection of the Pygopus activity. Avariety of such detection means may be used, including but not limitedto one or a combination of the following: radiolabelling (e.g. ³²P),antibody-based detection, fluorescence, chemiluminescence, spectroscopicmethods (e.g. generation of a product with altered spectroscopicproperties), various reporter enzymes or proteins (e.g. horseradishperoxidase, green fluorescent protein), specific binding reagents (e.g.biotin/(streptavidin), and others. Binding may also be analysed usinggenerally known methods in this area, such as electrophoresis on nativepolyacrylamide gels, as well as fusion protein-based assays such as theyeast 2-hybrid system or in vitro association assays, orproteomics-based approachs to identify Pygopus binding proteins.

The assay may be carried out in vitro utilizing a source of Pygopuswhich may comprise naturally isolated or recombinantly produced Pygopus,in preparations ranging from crude to pure. Recombinant Pygopus may beproduced in a number of prokaryotic or eukaryotic expression systemswhich are well known in the art. Such assays may be performed in anarray format. In certain embodiments, one or a plurality of the assaysteps are automated.

A homolog, variant and/or fragment of Pygopus which retains activity,particularly the ability to activate Wnt-responsive genes or inhibitcancer cell proliferation, may also be used in the methods of theinvention. Homologs includes protein sequences which are substantiallyidentical to the amino acid sequence of a Pygopus, sharing significantstructural and functional homology with a Pygopus. Variants include, butare not limited to, proteins or peptides which differ from a Pygopus byany modifications, and/or amino acid substitutions, deletions oradditions. Such variants include fusion proteins, for example a proteinof interest or portion thereof fused with a suitable fusion domain (suchas glutathione-S-transferase fusions, and others). Modifications canoccur anywhere including the polypeptide backbone, (i.e. the amino acidsequence), the amino acid side chains and the amino or carboxy termini.Such substitutions, deletions or additions may involve one or more aminoacids. Fragments include a fragment or a portion of Pygopus or afragment or a portion of a homolog or variant of Pygopus.

The assay may in an embodiment be performed using an appropriate hostcell as a source of Pygopus. Such a host cell may be prepared by theintroduction of DNA encoding Pygopus into the host cell and providingconditions for the expression of Pygopus. Such host cells may beprokaryotic or eukaryotic, bacterial, yeast, amphibian or mammalian.

The above-described assay methods may further comprise determiningwhether any compounds so identified can be used for the prevention ortreatment of cancer, such as examining their effect(s) on diseasesymptoms in suitable cancer animal model systems. The above-mentionedmethods may similarly be used to identify and characterize compounds forthe modulation of Pygopus in a system.

VI. Binding Assays

Pygopus can be used in assays related to the functional informationprovided herein; to raise antibodies or to elicit another immuneresponse; as a reagent (including the labeled reagent) in assaysdesigned to quantitatively determine levels of the protein in biologicalfluids; and as markers for tissues in which the corresponding protein ispreferentially expressed (either constitutively or at a particular stageof tissue differentiation or development or in a disease state). Wherethe protein binds or potentially binds to another protein or ligand, theprotein can be used to identify the binding partner/ligand so as todevelop a system to identify inhibitors of the binding interaction. Anyor all of these uses are capable of being developed into reagent gradeor kit format as commercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The Pygopus proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to Wnt-signalling and tocancer. Such assays involve Pygopus functions or activities orproperties useful for diagnosis and treatment of cancer, particularly incells and tissues that express Pygopus. Experimental data as providedherein indicates that Pygopus is over-expressed in human tumour tissue.

Pygopus and fragments, particularly the NHD region, are also useful indrug screening assays, in cell-based or cell-free systems. Cell-basedsystems can be native, i.e., cells that normally express Pygopus, as abiopsy or expanded in cell culture. In an alternate embodiment,cell-based assays involve recombinant host cells expressing Pygopus orits fragments, particularly the NHD region.

Pygopus and fragments, particularly the NHD region, can be used toidentify compounds that modulate the Wnt-signalling activation functionof the protein in its natural state or an altered form. Pygopus andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to Pygopusand fragments, particularly the NHD region. These compounds can befurther screened against a functional Pygopus to determine the effect ofthe compound on Pygopus activity. Further, these compounds can be testedin animal or invertebrate systems, particularly Xenopus, or in cancercell lines, to determine activity/effectiveness. Compounds can beidentified that activate (agonist) or inactivate (antagonist) Wntsignalling to a desired degree.

Further, Pygopus and fragments, particularly the NHD region, can be usedto screen a compound for the ability to stimulate or inhibit interactionbetween Pygopus and a molecule (binding partner) that normally interactswith Pygopus, e.g. Bcl-2. Such assays typically include the steps ofcombining Pygopus and fragments, particularly the NHD region, with atest compound under conditions that allow Pygopus and fragments,particularly the NHD region, to interact with the binding partner, andto detect the formation of a complex between Pygopus and the bindingpartner, or to detect the biochemical consequence of the interaction,such as activation of Wnt signalling.

Test compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries and combinatorial chemistry-derived molecularlibraries made of D- and/or L-configuration amino acids; 2)phosphopeptides (e.g., members of random and partially degenerate,directed phosphopeptide libraries); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′).sub.2, Fab expression libraryfragments, and epitope-binding fragments of antibodies); and 4) smallorganic and inorganic molecules (e.g., molecules obtained fromcombinatorial and natural product libraries).

Binding and/or activating compounds can also be screened by using fusionproteins in which the amino terminal domain, or parts thereof, and thecarboxy terminal domain, or parts thereof, can be replaced byheterologous polypeptides. These are generally referred to as chimericor fusion proteins.

Chimeric and fusion proteins comprise Pygopus and fragments,particularly the NHD region, operatively linked to a heterologousprotein having an amino acid sequence not substantially homologous tothe Pygopus in the fusion. “Operatively linked” indicates that thePygopus in the fusion and the heterologous protein are fused in-frame.The heterologous protein can be fused to the N-terminus or C-terminus ofthe Pygopus in the fusion.

In some uses, the fusion protein does not affect the activity of thePygopus in the fusion. For example, the fusion protein can include, butis not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant Pygopus and fragments, particularly the NHD region. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of a protein can be increased by using a heterologous signalsequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence. Moreover, manyexpression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A nucleic acid encoding Pygopus andfragments, particularly the NHD region, can be cloned into such anexpression vector such that the heterologous moiety is linked in-frameto Pygopus.

Pygopus and fragments, particularly the NHD region, are also useful incompetition binding assays in methods designed to discover compoundsthat interact with Pygopus (e.g. binding partners and/or ligands). Thus,a compound is exposed to Pygopus and fragments, particularly the NHDregion, under conditions that allow the compound to bind or to otherwiseinteract with the polypeptide. A known binding partner such as Bcl-9 oran antibody, particularly a monoclonal antibody to Pygopus, is alsoadded to the mixture. If the test compound interacts with Pygopus, itmay decrease the amount of complex formed between Pygopus and the knownbinding partner. This type of assay is particularly useful in cases inwhich compounds are sought that interact with specific regions ofPygopus. Thus, the binding partner that competes with the test compoundis designed to bind to peptide sequences corresponding to the region ofinterest in Pygopus.

In one embodiment, the competitor is a binding partner known to bind toPygopus, such as an antibody, peptide, ligand, etc. Under certaincircumstances, there may be competitive binding as between the testcompound and the binding partner, with the binding moiety displacing thecandidate agent.

Competitive screening assays may be done by combining Pygopus andfragments, particularly the NHD region, and a test compound in a firstsample. A second sample comprises the test compound, Pygopus andfragments, particularly the NHD region, and a known binding partner. Thebinding of the binding partner with Pygopus is determined for bothsamples, and a change, or difference in binding between the two samplesindicates the presence of an agent capable of binding to Pygopus andpotentially modulating its activity, i.e its interaction with the knownbinding partner. That is, if the binding of the candidate agent isdifferent in the second sample relative to the first sample, the testcompound is capable of binding to Pygopus.

In one embodiment, the test compound is labeled. Either the testcompound, or the binding partner, or both, is added first to Pygopus andfragments, particularly the NHD region, for a time sufficient to allowbinding. Incubations may be performed at any temperature whichfacilitates optimal activity, typically between 4 and 40 degree C.Incubation periods are selected for optimum activity, but may also beoptimized to facilitate rapid high throughput screening. Typicallybetween 0.1 and 1 hour will be sufficient. Excess reagent is generallyremoved or washed away. The second component is then added, and thepresence or absence of the labeled component is followed, to indicatebinding.

In a preferred embodiment, the binding partner is added first, followedby the test compound. Displacement of the binding partner is anindication the test compound is binding to Pygopus and thus is capableof binding to, and potentially modulating, the activity of Pygopus. Inthis embodiment, either component can be labeled. Thus, for example, ifthe binding partner is labeled, the presence of label in the washsolution indicates displacement by the test compound. Alternatively, ifthe test compound is labeled, the presence of the label on the supportindicates displacement.

In an alternative embodiment, the test compound is added first, withincubation and washing, followed by the binding partner. The absence ofbinding by the binding partner may indicate the test compound is boundto Pygopus with a higher affinity. Thus, if the test compound islabeled, the presence of the label on the support, coupled with a lackof binding by the binding partner, may indicate the test compound iscapable of binding to Pygopus.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either Pygopus and fragments, particularly the NHD region, orits binding partner, to facilitate separation of complexes fromuncomplexed forms of one or both components, as well as to accommodateautomation of the assay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads or glutathione derivatized microtitreplates, which are then combined with the cell lysates (e.g., 35S-labeled) and the candidate compound, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads are washedto remove any unbound label, and the matrix immobilized and radiolabeldetermined directly, or in the supernatant after the complexes aredissociated. Alternatively, the complexes can be dissociated from thematrix, separated by SDS-PAGE, and the level of Pygopus-binding proteinfound in the bead fraction quantitated from the gel using standardelectrophoretic techniques. For example, either the polypeptide or itstarget molecule can be immobilized utilizing conjugation of biotin andstreptavidin using techniques well known in the art. Alternatively,antibodies reactive with the protein but which do not interfere withbinding of the protein to its target molecule can be derivatized to thewells of the plate, and the protein trapped in the wells by antibodyconjugation. Preparations of a binding partner and a test compound areincubated in the Pygopus2-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodies, aswell as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the complex.

Agents that modulate or inhibit Pygopus and fragments, particularly theNHD region, can be identified using one or more of the above assays,alone or in combination. It is generally preferable to use a cell-basedor cell free system first and then confirm activity in an animal orother model system. Such model systems are well known in the art and canreadily be employed in this context.

In yet another aspect of the invention, Pygopus and fragments,particularly the NHD region, can be used as “bait proteins” in atwo-hybrid assay or three-hybrid assay to identify other proteins, whichbind to or interact with Pygopus and fragments, particularly the NHDregion. Such binding proteins are likely to be involved in thepropagation of signals of the Wnt pathway. Alternatively, such bindingproteins are also likely to be inhibitors of Pygopus.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for Pygopus andfragments, particularly the NHD region, is fused to a gene encoding theDNA binding domain of a known transcription factor (e.g., GAL-4). In theother construct, a DNA sequence, from a library of DNA sequences, thatencodes an unidentified protein (“prey” or “sample”) is fused to a genethat codes for the activation domain of the known transcription factor.If the “bait” and the “prey” proteins are able to interact, in vivo, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with Pygopus andfragments, particularly the NHD region.

This invention further pertains to agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., Pygopus-modulating agent, an antisense Pygopusnucleic acid molecule, a Pygopus-specific antibody, or a Pygopus-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

Pygopus and fragments, particularly the NHD region, are also useful toprovide a target for diagnosing cancer or predisposition to cancer.Accordingly, the invention provides methods for detecting the presence,or levels of, the protein (or encoding mRNA) in a cell, tissue, ororganism. Experimental data as provided herein indicates Pygopusover-expression in humans in various tumour tissues. The method involvescontacting a biological sample with a compound capable of interactingwith Pygopus and fragments, particularly the NHD region, such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

Pygopus and fragments, particularly the NHD region, also provide targetsfor diagnosing active protein activity, disease, or predisposition todisease. Thus, Pygopus can be isolated from a biological sample andassayed for the presence of a genetic mutation that results in aberrantprotein. This includes amino acid substitution, deletion, insertion,rearrangement, (as the result of aberrant splicing events), andinappropriate post-translational modification. Analytic methods includealtered electrophoretic mobility, altered tryptic peptide digest,altered activation of Wnt-signalling activity in cell-based or cell-freeassay, alteration in substrate or antibody-binding pattern, alteredisoelectric point, direct amino acid sequencing, and any other of theknown assay techniques useful for detecting mutations in a protein. Suchan assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

In vitro techniques for detection of Pygopus or fragments include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the proteincan be detected in vivo in a subject by introducing into the subject alabeled antibody or other types of detection agent. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.Particularly useful are methods that detect the allelic variant of apeptide expressed in a subject and methods which detect fragments of apeptide in a sample.

Pygopus and fragments, particularly the NHD region, are also useful incancer-related pharmacogenomic analysis. Pharmacogenomics deal withclinically significant hereditary variations in the response to drugsdue to altered drug disposition and abnormal action in affected persons.The clinical outcomes of these variations result in severe toxicity oftherapeutic drugs in certain individuals or therapeutic failure of drugsin certain individuals as a result of individual variation inmetabolism. Thus, the genotype of the individual can determine the way atherapeutic compound acts on the body or the way the body metabolizesthe compound. Further, the activity of drug metabolizing enzymes effectsboth the intensity and duration of drug action. Thus, thepharmacogenomics of the individual permit the selection of effectivecompounds and effective dosages of such compounds for prophylactic ortherapeutic treatment based on the individual's genotype. The discoveryof genetic polymorphisms in some drug metabolizing enzymes has explainedwhy some patients do not obtain the expected drug effects, show anexaggerated drug effect, or experience serious toxicity from standarddrug dosages. Polymorphisms can be expressed in the phenotype of theextensive metabolizer and the phenotype of the poor metabolizer.Accordingly, genetic polymorphism may lead to allelic protein variantsof Pygopus-2 in which one or more of the Pygopus-2 functions in onepopulation is different from those in another population. Accordingly,dosage of a certain drug may be modified to maximize the therapeuticeffect within a given population containing a polymorphism. As analternative to genotyping, specific polymorphic peptides could beidentified.

VII. Antibodies

The invention also provides antibodies that selectively bind to Pygopusas well as variants and fragments thereof (the target peptide). As usedherein, an antibody selectively binds a target peptide when it binds thetarget peptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target such that there are sharedepitopes. In this case, it would be understood that antibody binding tothe peptide is still selective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven antigen. Antibodies are generated by immunization of a mammal withPygopus or a fragment thereof. Such antibodies may be polyclonal ormonoclonal. Methods to produce polyclonal or monoclonal antibodies arewell known in the art. For a review, see “Antibodies, A LaboratoryManual, Cold Spring Harbor Laboratory, Eds. E. Harlow and D. Lane(1988), and D. E. Yelton et al., 1981. Ann. Rev. Biochem. 50:657-680.For monoclonal antibodies, see Kohler & Milstein (1975) Nature256:495-497.

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The antibodies may then be harvested from the organism.The full-length protein, an antigenic peptide fragment or a fusionprotein can be used. Particularly important fragments are those coveringfunctional domains, such as the NHD domain, and regions unique toPygopus or fragments thereof, such as those that can readily beidentified using protein alignment methods and as presented herein.

In one embodiment, a monoclonal antibody is produced which bindsspecifically to Pygopus or fragments thereof. The method comprisesimmunizing an animal with Pygopus or fragments thereof to produceimmunocytes for harvesting; obtaining immunocytes, such as splenocytes,from the immunized animal; and fusing the immunocytes with myeloma cell,whereby screening for fused cells identifies hybridomas which producethe monoclonal antibody.

Antibodies are preferably prepared from regions or discrete fragments ofPygopus or fragments thereof. Antibodies can be prepared from any regionof the peptide as described herein. However, preferred regions willinclude those unique to Pygopus, such as regions outside of the N-boxand the PHD domain.

An immunogenic epitope will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness.

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³I, ³⁵S or³H.

VIII. Antibody Uses

The antibodies can be used to isolate Pygopus or fragments thereof bystandard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment.

Experimental data as provided herein indicates that Pygopus isover-expressed in humans in various tumour tissues. Such antibodies canbe used to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Detection of Pygopus overexpression can be used to diagnosecancer. Such antibodies can also be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of cancer or in an individual with apredisposition toward disease related to the protein's function. When adisorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of Pygopus or fragments thereof to abinding partner such as Bcl-9. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) Pygopus activity. Antibodies canbe prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means fordetermining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

EXAMPLE 1 Pygopus is a Diagnostic and Therapeutic Target in EpithelialOvarian Carcinoma

(1) Detailed Protocols

(a) Cell culture: Cell lines were obtained from American Type CultureCollection (ATCC, USA) and were cultured in DMEM (GIBCO BRL, USA)containing 10% Fetal calf serum (FCS), 100 U/ml of penicillin, 100 μg/mlof streptomycin. Normal ovarian surface epithelial (OSE) cells werescraped from ovaries (patient consent approval) and cultured in MCDB105(Sigma, USA) and 199 (Sigma, USA) (1:1) containing 15% FCS, 100 U/ml ofpenicillin and 100 μg/ml of streptomycin as described (Wong, A. S. &Auersperg, N. Ovarian surface epithelium: family history and earlyevents in ovarian cancer. Reprod. Biol. Endocrinol. 1, 70 (2003)). Cellswere counted using a hemacytometer. TABLE 1 Characteristics of variousepithelial ovarian cell (EOC) lines EPITHELIAL OVARIAN CELL LINES TissueOVCAR-3 Ovary; epithelial; adenocarcinoma OV-90 Ovary; metastatic site:ascites malignant papillary serous adenocarcinoma TOV-21G Ovary; clearcell carcinoma ES-2 Ovary; clear cell carcinoma TOV-112D Ovary;endometrioid carcinoma SKOV-3 Ovary; metastatic site: ascitesadenocarcinoma OSE-2 Ovary; epithelial, normal IOSE-397 Ovary;epithelial, immortalized(b) RNA extraction and Northern Blotting: Total cellular RNA wasextracted using RNeasy Mini Kit (Qiagen, QP, CANADA). Northern blotanalysis was performed using a ³²P-dCTP-labelled cDNA probes inRapid-Hyb buffer at 65° C. for one hour and washed to high stringency in0.1× at 65° C. for 15 minutes as described (Lake, B. B. & Kao, K. R.Pygopus is required for embryonic brain patterning in Xenopus. Dev.Biol. 261, 132-148 (2003)).(c) Protein extraction and Western blotting: For protein extraction,80-90% confluent transfected or untransfected cell monolayers werewashed with cold Phosphate Buffered Saline (PBS), lysed immediately in2× Sodium Dodecyl Sulfate (SDS) loading buffer, passed through 21Gsyringe needle to shear the viscous DNA and loaded onto 10-12%SDS-denaturing polyacrylamide gel. The fractionated proteins were thentransferred to Hybond enhanced chemiluminescent (ECL) nitrocellulosemembrane (Amersham Pharmacia Biotech, PQ, CANADA) under semi-drycondition. Immunodetection was performed using the ECL system (AmershamPharmacia Biotech, PQ, CANADA). Anti-hPygo2 polyclonal antisera wereraised in New Zealand White rabbits as described (Reynolds et al.Developmental expression of functional GABAA receptors containing thegamma 2 subunit in neurons derived from embryonal carcinoma (P19) cells.Brain Res. Mol. Brain Res. 35, 11-18 (1996)) using an in vitrosynthesized polypeptide corresponding to amino acid residues 89-328,lacking both NHD and PHD conserved (Kramps, T. et al. Wnt/winglesssignaling requires BCL9/legless-mediated recruitment of pygopus to thenuclear beta-catenin-TCF complex. Cell 109, 47-60 (2002)) regions ofhPygo2, which was generated by subcloning hPygo2 cDNA sequences derivedby PCR from a human EST clone (I.M.A.G.E.) into pGex4T1 (Amersham) andtested by immunoprecipitation and western analysis. Anti β-Catenin, antic-Myc and anti Erk-1 antibodies were purchased from Santa Cruz. AntiGSK-3β and anti phospho-GSK-3α/β antibodies were purchased from CellSignaling Technology.(d) Immunohistochemistry: Archived, paraffin embedded tumors weresectioned at 5 microns, mounted on glass slides (Surgipath) andprocessed using a citrate-based antigen recovery protocol (Rorke, S.,Murphy, S., Khalifa, M., Chernenko, G., & Tang, S. C. Prognosticsignificance of BAG-1 expression in nonsmall cell lung cancer. Int. J.Cancer 95, 317-322 (2001)) with hPygo2 primary antisera (diluted 1000×)β-catenin mouse monoclonal primary antibodies (Santa Cruz, diluted2000×), anti-mouse and anti-rabbit HRP-linked secondary antibodiesdiluted 1/250× (Amersham), counterstained with hematoxylin and mountedin Permount (Fisher) as described (supra). The concentration of primaryantibody used was determined by level at which there was no staining,either background or non-specific observed using both pre-immune serumand secondary antibody alone.(e) Immunofluorescence: Cells were plated onto glass chamber slides andfixed in 4% paraformaldehyde for 30 minutes, rinsed in Phosphatebuffered saline (PBS) twice, followed by 0.2% triton-X100 in PBS (tPBS)for 10 minutes. Cells were blocked in 10% normal donkey or goat serumand incubated overnight at 4° C. with primary antibodies in 1.5% normalserum in PBS. The next day the slides were washed for 30 to 40 minutesin 0.2% tPBS and incubated for 30 minutes at room temperature withbiotinylated donkey anti-rabbit (Amersham) or Cy3 donkey anti-mouse(Jackson ImmunoResearch Laboratories, Inc.) secondary antibodies in 1.5%normal sera. After a 30 to 40 minute wash in 0.1-0.2% tPBS, cells wereincubated in streptavidin fluorescein (Amersham) in 1.5% normal sera/PBSfor 30 minutes, washed in 0.1-0.2% tPBS 30 to 40 minutes mounted in 10%glycerol/PBS or Vectashield (Vector Laboratories, Inc.) and viewed underconfocal microscopy using filters optimized for fluorescein and Cy3.

(f) Knockdown Assays: Antisense oligonucleotides were designed againsthPygo2 (Table 2). TABLE 2 Antisense oligonucleotides against hPygo2Oligo Target region name Sequence (5′-3′) (SEQ ID NO: 1) Hpy1GAGCTGCAGCAACCACAAAG 55-74 (SEQ ID NO: 5) Hpy2 GGACCCGGGTTAGCGGCAGCG144-164 (SEQ ID NO: 6) Hpy3 CCACCTCCCTCCAGCTTGTCC 198-219 (SEQ ID NO: 7)Hpy4 GGAGGACTAAAGTTTTGAC 687-705 (SEQ ID NO: 8) Hpy5 GGCTGAGCAAATCGTTGGG807-825 (SEQ ID NO: 9) Hpy6 GAAAAGCAGTAGAAGCAGGT 967-986 (SEQ ID NO: 10)Hpy7 CTCACGGATGTAGACAGA 1340-1357 (SEQ ID NO: 11) Hpy8CCTCTGGCCAGAAACCTTT 1817-1835 (SEQ ID NO: 12) Hpy9 CTCTTCTACCTTTGAGTAC2434-2452 (SEQ ID NO: 13) Hpy10 CACTGTATCTTGAGCTGG 2720-2737 (SEQ ID NO:14)

5×10⁴ of SK-OV-3 cells and 6×10⁴ of NIH-OVCAR-3 cells were seeded into12-well plates 24 hrs before transfection. 100-200 nM of 19 meroligonucleotides containing 3 phosphorothioated bonds (*) at eachterminus (5′-G*G*C*TGAGCAAATCGTT*G*G*G-3′; Hpy5) from the coding region(nt no. 807-825) of hPygo2 (SEQ ID NO:1) and its mismatched sequence(5′-G*C*C*TGAGCTAATCATT*G*G*T-3′; SEQ ID NO:20) or anti-Xenopus pygo2oligonucleotides (5′-T*T*T*GCGCCGTTTCTT*C*T*C-3′; SEQ ID NO:21) wastransfected into NIH-OVCAR-3 using the Oligofectamine Transfection Kit(Invitrogen, CA, USA) and SK-OV-3 using Effectene Transfection Kit(Qiagen, QP, CANADA). The culture medium was changed after 24 and 48hours transfection.

β-Catenin siRNA, consisting of 4 pooled siRNAs was purchased fromUpstate. hPygo2 siRNA was chemically synthesized from Qiagen. IndividualsiRNAs were tested for their ability to knock down hPygo2 as assayed bywestern analysis. Hpy2A: 5′-r(CGAUGACCAGGAUGCCAUU)d(TT)-3′ Hpy2B:5′-r(AGAAGCGAAGGAAGUCAAA)d(TT)-3′ Hpy2C:5′-r(UGGGAACCAGCCCAGUUUC)d(TT)-3′ Hpy2D:5′-r(CCAGCCUCUGGGUCAAAAC)d(TT)-3′ Hpy2E:5′-r(CUUUCCCAGCCAACCCUUC)d(TT)-3′

Two (Hpy2A and Hpy2D) out of 5 of the siRNAs were effective in knockingdown hPygo2.

Transfection of siRNA was performed as for the antisense ON, usingeither Oligofectamine or RNAiEasy (Qiagen). Cells were washed in PBS 6hours after transfection of 100 nM of siRNA and replaced with freshmedium, followed by an additional transfection of 100 nM of siRNA 24hours later. The cells were then fixed and stained with propidium iodideand counted for DNA content using a Fluorescence Activated Cell Sorter.

(2) Specificity of Anti-hPygo2 Antibody

Different regions of hPygo2 were fused to Gal-4 and the constructs weretransiently transfected into HeLa cells. Protein was extracted andanalyzed for the expression of the transfected constructs. The sameamount of protein was loaded and transferred for both blots. FIG. 3(a)depicts Gal-4-hPygo2 fusion protein constructs that were transfectedinto HeLa cells. FIG. 3(b) shows a Western blot of Gal-4-hPygo2constructs using anti-hPygo2 antiserum; see Example 1(1). FIG. 3(c)shows a Western blot of Gal-4-hPygo2 constructs using an anti-Gal-4antibody. The Gal-4 antibody recognizes all Gal-4-hPygo2 proteinconstructs.

FIGS. 3(d) and (e) show similar experiments conducted using fusions toFlag peptide.

FIG. 3(f) depicts immunocytochemical analyses of four OvCa cell linesusing anti-hPygo2 antibodies and beta-catenin. The results show that allfour lines express hPygo but only two express beta-catenin. Thus Pygopusis more closely correlated with malignant epithelial ovarian cancercells than other wnt signaling components.

(3) Overexpression of hPygo2 in EOC Cell Lines

We initially surveyed β-catenin and GSK-3β expression in six malignantEOC cell lines (OV-90, NIH-OVCAR-3, TOV-21G, TOV-112D, SK-OV-3 and ES-2)to determine the role of canonical Wnt signaling in this malignancy(FIG. 4 a). β-catenin was overexpressed in TOV-112D cells butunderexpressed or not expressed in the other lines, relative to normalovarian surface epithelial (OSE) cells. Also, only two EOC lines, TOV21Gand SKOV-3, expressed the activated, phosphorylated form of GSK3-β.Thus, β-catenin expression and that of its regulatory factor, GSK3-β isvariable in these EOC cell lines.

Deregulation of the Wnt pathway in cancer results largely frominactivating mutations in genes encoding β-catenin regulatory factors,or activating mutations in β-catenin itself. In either case, the resultof pathway hyperstimulation is an increase in expression of target genesinvolved in cell cycle progression, including cyclin D1 and c-myc. Wedid not, however, find consistent coordinate expression of β-cateninwith these targets across the EOC lines (FIG. 4 a). This lack ofcorrelation in the expression of Wnt pathway components upstream anddownstream of Pygopus does not support a general role for canonical Wntsignaling in EOC, so we determined whether this inconsistency also wastrue for hPygo2. In contrast to the other components, hPygo2 mRNA andprotein (FIG. 4 b) were overexpressed in every EOC cell line weexamined. The levels of hPygo2 mRNA were significantly higher in thecancer cell lines relative to that of normal surface epithelial cells.In addition, while hPygo2 protein was very highly expressed in thecancer cell lines, it was undetectable in the normal cells. Theseobservations suggested that overexpression of Pygopus is acharacteristic of EOC.

(4) Pygopus, but not β-catenin is Consistently Over-Expressed in PatientTumors

To assess the involvement of Pygopus in disease, we determined the insitu expression of hPygo2 protein in 125 tumors from archived surgicalsamples. We semi-quantitatively assessed expression based on intensityand percentage of tumor cells stained in both the cytoplasm and nuclei(FIG. 4 c). We looked at all EOC tumor subtypes, including serous,mucinous, clear cell, endometrioid, and undifferentiated but themajority, as expected, were of the serous subtype. hPygo2 was notexpressed in the nuclei of benign tumors such as endometriosis (data notshown) or was very weakly expressed in non-invasive tumors such asbenign cystadenomas (FIG. 4 c). On the other hand, 82% of the tumorswith a pathological diagnosis consistent with malignant EOC had moderateto strong nuclear accumulation of hPygo2 protein (Table 3) and thestaining occurred exclusively in tumor cells but not in the surroundingstroma. Fewer EOC tumors displayed strong cytoplasmic staining and therewas no preferential expression of hPygo2 in any tumor subtype. Theseanalyses are consistent with a role for Pygopus in malignant disease.

We also compared β-catenin expression with hPygo2 in adjacent sectionsfrom 87 of the 125 tumor specimens from patients diagnosed withmalignant EOC collected above (FIG. 4 c, Table 4). Eighty-four, or 97%of the tumors stained positive for nuclear hPygo2. Of these, only 8 (9%)of the tumors also stained for nuclear β-catenin. More than half (56%),or 49 of the tumors stained positive for cytoplasmic β-catenin and ofthese, 47 (54%) stained positive for nuclear hPygo2. None of the tumorsstained exclusively for nuclear β-catenin while only 2 tumors hadcytoplasmic P-catenin staining without any hPygo2 staining. Only onetumor did not stain for either protein. These observations indicatedthat the high frequency of excess nuclear accumulation of hPygo2 islargely unaccompanied by nuclear staining of β-catenin in EOC tumors.

(5) Antisense Oligonucleotides and siRNA Specifically Target hPygo2 forKnockdown

The high frequency of expression of hPygo2 we observed relative toβ-catenin in tumors and cell lines, suggested that Pygopus has a moregeneralized role in EOC and may therefore be an important therapeutictarget.

We used phosphorothioated antisense oligonucleotides (ON) to knockdownhPygo2 in EOC cell lines. We identified antisense oligonucleotidescapable of knocking down hPygo2 expression. FIG. 3(g) maps antisenseoligonucleotides designed against the full hPygo2 cDNA sequence,avoiding conserved sequences such as the NHD and PHD domains. In FIG.3(h), antisense oligonucleotides were transfected into HeLa cells at aconcentration of 250 nM. RNA was extracted 24 hours later and RT-PCRanalysis was performed to assess the relative knockdown of hPygo2 RNAlevels. Densitometry was performed, standardizing the relative hPygo2levels to the relative GAPDH levels. RT−, negative control, withoutreverse transcriptase. TABLE 3 Immunohistochemical nuclear andcytoplasmic staining of hPygo2 in EOC tumors distributed by tumorsubtype. Numbers in parentheses indicate percentages of totals shown inbottom row. Tumor Subtype Endome- Undiffer- Benign Mucinous trioidSerous Clear cell entiated Total Nuclear − 7(100)  3(21) 0  2(2) 0 0 12(10) + 0  1(7) 1(12)  8(9) 0 0  10(8) ++ 0  7(50) 5(63) 46(53) 4(80)3(75)  65(52) +++ 0  3(21) 2(25) 31(36) 1(20) 1(25)  38(30) Cytoplasmic− 5(72)  4(29) 0 12(14) 0 0  21(17) + 0  6(43) 5(63) 53(61) 5(100) 1(25) 70(56) ++ 1(14)  3(21) 3 21(24) 0 2(50)  30(24) +++ 1(14)  1(7) 0(37) 1(1) 0 1(25)  4(3) Total 7(100) 14(100) 8(100) 87(100) 5(100) 4(100)125(100)

TABLE 4 Distribution of EOC tumors based on nuclear and cytoplasmicβ-catenin staining in relation to nuclear staining of hPygo2. NuclearhPygo2 − + ++ +++ Total Nuclear β-catenin − 3 8 43(90) 25(93) 79(91) + 01  4(8)  1(4)  6(7) ++ 0 0  1(3)  1(4)  2(2) Cytoplasmic β-catenin − 1 422(46) 11(41) 38(44) + 2 3 21(44) 11(41) 37(43) ++ 0 2  5(10)  5(19)12(14) Total number of tumors 3 9 48(100) 27(100) 87(100)β-Catenin staining was assessed as either negative (−), weak (+) ormoderate to strong (++). Numbers in parentheses indicate percent oftotals shown in bottom row.

Of the 10 Ons spanning hPygo2 and the eight ONs spanning the length ofthe coding region of hPygo2 (Table 2; FIGS. 3(g) and (h)), threesignificantly knocked down endogenous hPygo2 (Hpy5, Hpy8 and Hpy10), themost effective one of which we used for experiments (Hpy5).

Both hPygo2 mRNA and protein levels were significantly reduced by theantisense ON as compared to controls, without affecting the expressionof hPygo1 RNA or β-catenin protein (FIG. 5 a). Of the two cell linestested, the ON reduced endogenous hPygo2 protein by about 40% in SK-OV-3cells while there was a reduction by approximately 30% of the endogenoushPygo2 level in OVCAR-3 cells (FIG. 5 a).

We next tested small interfering RNAs (siRNA) to provide an alternativemeans to knockdown hPygo2. Out of five siRNA sequences, two (Hpy2A andHpy2D) were effective in reducing hPygo2 levels to 50% (Hpy2A) and 32%(Hpy2D) of the mock transfected control levels in both OVCAR-3 andSK-OV3 cells (FIG. 5 b). This reduction was specific to hPygo2, as thelevels of β-catenin were not significantly different from that of cellstransfected with the negative control siRNA. The requirement ofβ-catenin was also tested using commercially available anti-β-cateninsiRNA, which caused a significant reduction of endogenous β-catenin toless than 10% in both cell lines, while not affecting the levels ofhPygo2. These experiments indicated that both antisense ON and siRNA areeffective ways to knockdown hPygo2 in EOC.

(6) hPygo2 and β-Catenin do not Colocalize

Based on our immunoblot data (FIG. 4 a), β-catenin is expressed in bothSKOV-3 cells and OVCAR-3 cells. Demonstration of nuclear co-localizationof hPygo2 with —catenin, therefore, would provide evidence that hPygo2is involved in canonical Wnt signaling in EOC cell lines. Studies usingconventional fluorescence microscopy may be confounded by highautofluorescence levels and low resolution associated with conventionalfluorescence microscopy. So we used confocal microscopy to unambiguouslyvisualize the normal expression and knockdown by ONs and siRNA of hPygo2and β-catenin in SK-OV-3 and OVCAR-3 EOC cell lines in situ (FIG. 6). Inthe untreated (FIG. 6 a, e,

i) and control transfected (FIG. 6 c, g) cells, β-catenin was localizedto the plasma membrane, whereas hPygo2 was always concentrated innuclei. In cells transfected with both antisense ONs and siRNA specificfor hPygo2, however, the concentration of hPygo2 in nuclei was noticablyreduced (FIG. 6 d, h, l). In these cases β-catenin was still oftenexpressed and continued to be localized at points of cell-to-cellcontact (FIG. 6 d, h). Interestingly, knockdown of β-catenin in theSK-OV-3 cells resulted in a slight dispersion of hPygo2 protein fromnuclei, but the cells clearly continued to overexpress hPygo2 (FIG. 6k). Thus, the lack of co-localization of hPygo2 and β-catenin suggeststhat the activities and functions of these proteins are not coupled inEOC cell lines.

(7) Requirement of hPygo2 for EOC Cell Survival

We established the cellular requirement for Pygopus after knockdown ofhPygo2 and β-catenin in SKOV-3 and OVCAR-3 cell lines. Significantly,both of these EOC lines underwent growth reduction 48 and 72 hours afterantisense hPygo20N transfection as compared to untreated cells and cellstransfected with control ONs (FIG. 7 a). hPygo2 siRNA also caused markedreduction in cell numbers of both cell lines 48 and 72 hourspost-transfection (FIG. 7 b). β-catenin siRNA, on the other hand, causeda significant reduction in OVCAR-3 but only a slight reduction inSK-OV-3 cell numbers. Interestingly, knockdown of β-catenin in SK-OV-3cells resulted in a modest reduction in hPygo2 and a consequent partialdecrease in cell numbers. Whether or not the effects on cell growth inthis instance are due to the loss of β-catenin or partial reduction inhPygo2 is unclear but it is possible that EOC cell survival is dependenton the expression of hPygo2, which in turn is partially dependent on Wntsignaling, consistent with previous findings that TCF/LEF is a Wnttarget.

The loss of EOC cell numbers could result either from thesiRNA-transfected cells undergoing cell death or from cell cycle arrest.Accumulation of cells with DNA content less than that of cells in G1,for instance, would signify loss of DNA, resulting from death, while areduction in cell numbers in M and S-phase would indicate growth arrest.To distinguish these possibilities, we processed cells in which hPygo2or β-catenin was knocked down, for fluorescence activated cell sorting(FACS) using propidium iodide to monitor DNA content (FIG. 7 b). After48 hours, untreated and control treated cells showed modest differencesin the distribution of cells in G1, S-phase and M-phase of the cellcycle. There was a significant increase in the fraction of sub G1 phasecells in both SKOV-3 and OVCAR-3 cells treated with hPygo2 siRNA.Consistent with the counting assays, we found that only the OVCAR-3cells were most sensitive to β-catenin siRNA, while the SKOV-3 cellstreated with β-catenin siRNA showed a modest increase in the proportionof sub G1 cells compared to the untreated controls. These findingsindicate that removal of hPygo2 from EOC cells causes them to die,indicating a role of Pygopus in EOC cell survival.

(8) Hypothesis on Mechanism

Pygopus was proposed to be dedicated to the canonical Wnt/β-cateninpathway but our results now indicate that it can also functionindependently of β-catenin in EOC. Pygopus either has non-Wnt activity,therefore, or its sole overexpression in cancer is sufficient toactivate Wnt target gene expression, making β-catenin dispensable,perhaps by causing localized chromatin remodeling effects either alone,or in combination with other partnering proteins. Other non-canonicalWnt components, such as Plakoglobin (y-catenin) can bind to TCF/LEF andtherefore might be recruited by Pygopus. Alternatively, Pygopus may beinvolved in other non-canonical Wnt processes such as the Wnt/Ca2+ andplanar cell polarity (PCP) pathways that are important forembryogenesis, but not yet defined in cancer. These alternatives,however, do not preclude the possibility that the interaction betweenoverexpressed Pygopus and other Wnt components may be still intact inthe absence of β-catenin and may be sufficient for Wnt target geneexpression.

We have found that hPygo2 knockdown suppresses the growth of breast(MCF-7), two additional ovarian (TOV-112D, TOV21G) and cervical (HeLa)cancer cell lines (see below) supporting the hypothesis that Pygopusplays a generalized role in malignancy.

The high frequency of expression of Pygopus in malignant tumors, coupledwith its requirement for EOC cell survival suggests that inhibition ofPygopus activity is a feasible strategy against cancer.

EXAMPLE 2 Pygopus is a Diagnostic and Therapeutic Target in BreastCancer, as Well as Ovarian and Cervical Cancer

(1) Detailed Protocols

(a) hPygo2 clones and antibody production: The region encoding aminoacids 89-328 of hPygo2 lacking both the NHD and PHD conserved sequenceswas PCR amplified (F: 5′-GCATCCAACCCTTTTGAAGATGAC SEQ ID NO:22; R:5′-TCAGCCAGGGGGTGCCAAGCTGTTG SEQ ID NO:23) from I.M.A.G.E. Consortium(LLNL) hPygo2 cDNA clones (CloneIDs: 41570072 and 3627860) obtained fromIncyte Genomics Inc. and ligated into pGEX-4T1 (Amersham). Purifiedproteins were synthesized and isolated from BL-21 (RIL) cells (a giftfrom Dr. G. Paterno) using the GST Gene Fusion System (Amersham), as permanufacturers protocol. Preimmune serum was collected from rabbits(Charles River Laboratories) which were subsequently injected with 500μg of purified GST-hPygo2 fusion protein resuspended in phosphatebuffered saline. Serum collection and boosting was performed asdescribed (Ryan P J and Gillespie L L. (1994). Dev Biol, 166, 101-111).

(b) Northern blotting and RT-PCR: Total RNA was extracted from celllines using the Nucleospin RNA II Kit (Clontech). Northern blot analysiswas performed as previously described (Lake and Kao 2003) usingradioactively labeled probes generated by random labeling (Prime-a-Gene;Promega) of the PCR product of hPygo2 used in antibody preparation.Blots were washed at high stringency (60° C. in 0.1% SDS and 0.1×SSC)and reprobed with GAPDH (pTR1-GAPDH; Ambion) under the samehybridization conditions.

Semiquantitative RT-PCR was performed as previously described (Lake B Band Kao K R. (2003). Dev Biol, 261, 132-148) using hPygo2oligonucleotide primers described above. Primers for hPygo1 (Kramps etal. (2002). Cell, 109, 47-60) (F: 5′-GCCACGACAACCAAGAGGTG SEQ ID NO:24;R: 5′-CCAGTACAGATCCGATGAAACC SEQ ID NO:25), Bcl-9 (Willis et al. (1998).Blood, 91, 1873-1881) (F: 5′-GATGTTGTCCTGGTGTCTTG SEQ ID NO:26; R:5′-GGTCACGACACTGCAGTGCTC SEQ ID NO:27) and GAPDH (Ju et al. (1995).Nature, 373, 444-448) were synthesized by Invitrogen.

(c) Western blotting: Monoclonal and polyclonal β-catenin antibodieswere purchased from Santa Cruz Biotechnology, monoclonal β-actinantibodies from Sigma, and Cyclin D1 monoclonal antibodies from BDBiosciences. Total protein from tissue culture cells was extracted inprotein sample buffer. As a positive control, in vitro translated hPygo2protein was prepared using the transcription/translation coupledcell-free system (Promega). Approximately 50 μg of total cell lysate wasseparated by SDS-PAGE, transferred to nitrocellulose membranes(Hybond-ECL™; Amersham) and visualized by enhanced chemiluminescence(Amersham). Blots were reprobed with β-actin to confirm equal loading ofprotein.

(d) Immunocytochemistry and Immunohistochemistry: For immunofluorescenceanalysis, Hs-574-mg, Bt-474 and Mcf-7 cells were fixed in 4%paraformaldehyde (30 minutes) rinsed in PBS twice and 0.2% triton-X100/PBS (tPBS) for 10 minutes. Cells were blocked in 10% normaldonkey/goat serum prior to an overnight incubation with primaryantibodies in 1.5% normal serum/PBS. After a 30 to 40 minute wash in0.2% tPBS, cells were incubated 30 minutes with secondary antibodies in1.5% normal sera. For hPygo-2, biotinylated donkey anti-rabbit(Amersham) and for β-Catenin, Cy3 donkey anti-mouse (JacksonImmunoResearch Laboratories, Inc.) was used. After a 30 to 40 minutewash in 0.1-0.2% tPBS, cells were incubated in streptavidin fluorescein(Amersham) in 1.5% normal sera/PBS for 30 minutes. Cells were washed in0.1-0.2% tPBS 30 to 40 minutes before mounting in 10% glycerol/PBS orVectashield (Vector Laboratories, Inc.). Images were collected usingconfocal microscopy (Olympus).

Immunohistochemistry was carried out essentially as previously described(Rorke et al. (2001). Int j Cancer, 95, 317-322). Breast tumor sectionswere obtained from the Memorial University Division of LaboratoryMedicine.

(e) Antisense oligos and siRNA: Antisense oligonucleotides (Invitrogen)against hPygo2 were designed to contain three phosphorothioate bonds ateach terminus as indicated by asterisks to enhance nuclease resistance.The sequences used were as follows: hPygo2 antisense oligo;5′-G*G*C*TGAGCAAATCGTT*G*G*G (Hpy5; SEQ ID NO:9), Xenopus Pygo2-specificcontrol oligo; 5′-T*T*T*GCGCCGTTTCTT*C*T*C SEQ ID NO:21, 4 base mismatcholigo; 5′-G*U*C*TGAGCUAATCATT*G*G*T (mismatches underlined; SEQ IDNO:28). All oligonucleotides were designed avoiding G quartets andrepeated CG sequences which may result in non-specific antisenseeffects. β-Catenin siRNA and non-specific control siRNA were purchasedas a β-Catenin siRNA/siAB™ Assay Kit (Upstate). hPygo2 siRNA wassynthesized using the (Xeragon-Qiagen) sense sequences: Hpy2A;5′-r(CGAUGACCAGGAUGCCAUU)dTT-3′ (SEQ ID NO: 15) Hpy2D;5′-r(CCAGCCUCUGGGUCAAAAC)dTT-3′. (SEQ ID NO: 18)(f) Cell culture and transfection: All cell lines, except normalendocervical (HEN) and normal ectocervical (HEC) cell lines (Tsutsumi etal. 1992), were purchased from the American Type Culture Collection.T98G and Sk-N-Sh cells were maintained in Minimal Essential Media(Gibco) supplemented with 10% fetal bovine serum. HEN and HEC cells weremaintained in Keratinocyte Serum Free Media (Gibco). All remaining cellswere maintained in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum (Gibco).

All transfections utilized Oligofectamine (Invitrogen) as per themanufacturer's instructions, replacing the growth media every 24 hours.hPygo2 antisense/control oligonucleotides were transfected to a finalconcentration of 250 nM and all siRNAs were transfected at a finalconcentration of 100 nM. siRNA was transfected again 24 hours after thefirst transfection. For RT-PCR analysis, cells were seeded at a densityof 1.5×10⁵ cells/well in six-well plates and were harvested 24 hoursafter transfection for RNA extraction. For western analysis cells wereseeded at a density of 10⁵ cells/well in twelve-well plates and wereharvested 48 hours after transfection. For cell growth analysis cellswere seeded in triplicate at a density of 7.5×10⁴ cells/well intwelve-well plates, and were counted 48 and 72 hours after transfectionusing trypan blue exclusion (Sigma) with a hemacytometer.

(2) Pygopus is Expressed in Malignant Breast Cancer

The expression of hPygo2 in archived surgical breast tumor specimens wasdetermined by immunohistochemical analysis using antiserum we developedto react with non-conserved domains of the hPygo2 protein. We obtained22 archived breast tumor sections and 1 normal breast section andstained them with hPygo2 (FIG. 8). In 14 tumors, there was staining ofhPygo2 in malignant cells, but not in the surrounding non-tumor cells.Of the 14 positively stained specimens, 6 had distinctnuclear/cytoplasmic hPygo2 staining, whereas 8 had cytoplasmic hPygo2staining. hPygo2 could not be detected in the normal breast tissuesection. We also obtained 4 lymph node sections from breast tumorpatients. Two out of four contained metastatic tumor cells which stainedpositive for hPygo2, the other two did not contain tumor cells and didnot stain with hPygo2. The consistent expression of hPygo2 in themalignant cells of these tumors suggested that Pygopus plays animportant role in breast cancer.

To study the role of Pygopus in malignant breast cancer cell lines, theexpression of hPygo2 mRNA was determined in the breast cancer cell linesBt-474 and Mcf-7 and compared to a variety of cell lines derived fromnormal tissues and other malignancies using northern blot analyses (FIG.9 a). Messages were highly expressed in both ovarian cancer (Sk-Ov-3,Es-2), cervical cancer (HeLa, CaSki), breast cancer cell lines (BT-474,Mcf-7), and normal ectocervical (HEC) cells. Alternatively, hPygo2 mRNAexpression was very low or absent in neuroblastoma (T98G, Sk-N-Sh),normal endocervical (HEN) and normal breast (Hs-574) cell line.

The hPygo2 protein migrates to the predicted size of approximately 50KDa (FIG. 9 b). Expression of hPygo2 protein in the cell lines wasconsistent with the expression of hPygo2 mRNA (FIG. 9 c), with higherexpression in ovarian, cervical, normal ectocervical and breast cancercells and lower expression in neuroblastoma, normal endocervical andnormal breast cells. Examination of βCatenin protein expressiondemonstrated that it very closely resembled that of hPygo2 in most ofthe cell lines (FIG. 9 c). Notably, hPygo2 expression was absent in thenormal breast cell line (Hs-574), but was expressed at very high levelsin two breast cancer cell lines (Bt-474, Mcf-7).

(3) β-Catenin and hPygo2 do not co-Localize in Mcf-7 and Bt-474 Cells

We next used indirect immunofluorescence and confocal microscopy todetermine the subcellular localization of Pygopus and β-Catenin innormal and malignant breast cell lines. Endogenous hPygo2 protein waspredominantly localized to nuclei of both breast cancer cell linesanalyzed (Bt-474 and Mcf-7) (FIG. 10). In contrast, hPygo2 waspredominantly localized to the cytoplasm of the normal breast cell line(Hs-574) (FIG. 10). Unexpectedly, unlike hPygo2, β-Catenin wasconsistently found in the cytoplasm and associated with the inner cellmembrane of the normal and cancer cell lines and only weakly in thenucleus. Therefore, Pygopus and β-Catenin showed a preferentiallocalization to different compartments in the breast cancer cells.

(4) Bcl-9 is not Expressed in Breast Cancer Cell Lines and does notCorrelate with Pygopus

The interaction of Pygopus proteins with β-Catenin was shown to bemediated by Legless/Bcl-9. The lack of correlation between hPygo2 andβ-Catenin subcellular localization, however, would predict that theWnt/β-Catenin complex is, unexpectedly, uncoupled from hPygo2 in breastcancer. Given that Pygopus interacts with the β-catenin complex throughBcl-9, we assessed the relative expression levels of Bcl-9 by RT-PCR ina variety of cell lines (FIG. 9 d). Bcl-9 mRNA was expressed highly inone cervical (HeLa), two ovarian (Sk-Ov-3, Es-2), and two neuroblastoma(T98G, Sk-N-Sh) cell lines, while it was expressed at lower levels inall the other cell lines examined. Surprisingly, there was littlecorrelation in the expression of Bcl-9 with the expression of hPygo2 andβ-Catenin. In addition, Bcl-9 was expressed at lower levels in thebreast cancer cell lines as compared to the normal breast cells. Theseresults suggest that the function of hPygo2 in breast cancer may notrequire its interaction with the β-catenin transcription complex.

(5) β-Catenin is not Required for Growth of MCF-7 Cells

It was previously shown that Mcf-7 cells exhibit Wnt dependenttranscription and expression of the Wnt target gene, Cyclin D1. Weanalyzed, therefore, the requirement of β-Catenin for cell growth inMcf-7 cells by knocking down its expression with siRNA. Knockdown ofβ-Catenin with siRNA resulted in a significant decrease of β-Cateninprotein, which was not detectable by immunoblotting (FIG. 11 a).However, this was accompanied by no apparent change in cell numberscompared to transfection reagent and non-specific siRNA controls (FIG.11 b). Protein levels of both hPygo2 and Cyclin D1 remained unchangedafter β-Catenin knockdown compared to a non-specific siRNA (FIG. 11 a).These results provide direct evidence that β-Catenin is not required forcell proliferation, nor the expression of the Wnt target gene Cyclin D1in Mcf-7 cells.

(6) Pygopus is Required for the Proliferation of Mcf-7 Cells

The lack of requirement of β-Catenin for growth of Mcf-7 cells isconsistent with our observation that it is predominantly localized tothe membrane compartment of Mcf-7 and Bt-474 cells (FIG. 10). On theother hand, since hPygo2 consistently localized to the nucleus it ispossible that, in Mcf-7 cells, its requirement for growth is distinctfrom a function associated with β-Catenin. To test this possibility, wedetermined the requirement for Pygopus in cell growth in RNA antisenseknockdown experiments. In preliminary experiments we designed a vectorencoding antisense RNA complementary to hPygo2 that was able to suppressexponential growth of HeLa cells. We also designed a number of antisenseONs specific to hPygo2 mRNA (FIG. 3). A single oligonucleotide (hpy5),which had the greatest ability to knock down hPygo2 mRNA expression, waschosen for further analysis in HeLa cells. We used two controls ONs, onecomplementary to Xenopus Pygo2 (Non-specific) and the othercomplementary to (hpy5) with a 4 base-pair sequence change (Mismatch).Specific knock down of hPygo2 mRNA expression 24 hrs after transfectionof (hpy5) compared to the control ONs was achieved without affecting theexpression of the alternate, but related Pygo family member hPygo1 (FIG.12 a). Hpy5 also knocked down endogenous hPygo2 protein to less than 50%of the controls (FIG. 12 b).

As in the HeLa cells, transfection of the (hpy5) ON into Mcf-7 cellsresulted in a significant knockdown in hPygo2 protein levels as comparedto the non-specific and mismatch control ONs while β-Catenin levelsremained unaltered (FIG. 13 a). Most significantly, there was aconsiderable reduction of Mcf-7 cell growth (FIG. 13 b) aftertransfection with the hPygo-specific ON, as compared to the cellstransfected with the non-specific and mismatch control ONs. Cell numberswere reduced to 52%, as compared to 93% with the non-specific controland 83% for the mismatch control as compared to the reagent(oligofectamine) control. These results demonstrate that the ability ofthe hpy5 oligonucleotide to knockdown hPygo2 protein is specific andresults in a reduction in cell proliferation. This reduction in cellnumber was accompanied by a decrease in the cell cycle regulatoryprotein Cyclin D1 (FIG. 13 a), implying that the reduction of cellgrowth is likely due to cell cycle arrest. These results demonstratethat hPygo2 is required independently of β-Catenin in the growth ofMcf-7 cells.

To confirm that knockdown of hPygo2 by antisense ON results in adecrease of Mcf-7 cell proliferation we used siRNAs specific for hPygo2.Two (Hpy2A and Hpy2D) out of six siRNAs designed against hPygo2 werefound to be effective in reducing hPygo2 protein levels (FIG. 14)without effecting β-Catenin protein levels, and were therefore used forfurther experimentation. Coincident with this reduction in proteinlevels, we found that both of the siRNAs alone or in combination, causeda significant reduction in Mcf-7 cell numbers (FIG. 14 b), thereforeconfirming our results with the antisense ON.

(7) Hypothesis on Mechanism

Our results demonstrate that Pygopus is overexpressed in breast andother tumors and is required for Mcf-7 cell growth by a mechanismindependent of β-Catenin. Together our observations suggest that Pygopusproteins may be overexpressed in cancer and may play an additional role,other than its previously suggested one in mediating the canonical Wntsignal through β-Catenin/Bcl-9.

Our results demonstrated that the expression levels of Pygopuscorrelated with the expression of β-Catenin but not its only knownbinding partner, Bcl-9. The breast cancer cell lines used in this study,were previously shown to exhibit Wnt dependent transcription, Cyclin D1expression, as well as β-Catenin/Tcf complex formation. Therefore, thelack of Bcl-9 expression in the breast cancer cells is unexpected, sinceBcl-9 is required to tether Pygopus to the β-Catenin/Tcf complex. It ispossible that Pygopus and Bcl-9 are transcriptionally controlled bydifferent regulatory factors and this is why we see differences in theexpression patterns.

Mutations in genes encoding components required for Wnt signaling occurat relatively low frequencies in breast cancer, but it has beenhypothesized that the overexpression of Wnt signaling components leadsto the overexpression of target genes, and thus may contribute tomammary carcinogenesis. For example, β-Catenin has been reported to havenuclear/cytoplasmic staining in approximately 60% of breast tumortissues in two independent studies. This phenomenon may be due to Wntindependent regulation of β-Catenin, such as the regulation of β-Cateninby fibroblast growth factor and epidermal growth factor family members,as well as the Wnt independent regulation of GSK3.

Interestingly, Pygopus mRNA and protein were highly expressed in thebreast cancer cell lines Bt-474 and Mcf-7 compared to Hs-574 normalbreast cells and may indicate a requirement for Pygopus in theirproliferation. Pygopus is also known to function as a nuclear protein;we have confirmed this by the examination of endogenous subcellularlocalization. Its concentration in the nuclei of tumor cells butcytoplasmic preference or absence in normal cells. This indicates thatPygopus overexpression and subcellular localization may play a role inmalignancy. The display of nuclear Pygopus expression in some patienttumors is consistent with this role.

It has previously has been shown that Wnt signaling, and expression ofthe Wnt target gene Cyclin D1 is at relatively high levels in Mcf-7cells compared to a number of other breast carcinoma cell lines.Because, as we have shown, β-Catenin is not required for the expressionof Cyclin D1 in these cells, the expression of Cyclin D1 may not be atrue indication of Wnt signaling. Indeed, Cyclin D1 expression has beenshown to be required for cell growth and regulated by other non-Wntdependent proteins in Mcf-7 cells, such as Estrogen receptor andPeroxisome Proliferator-activated Receptor γ(PPARγ). The majority ofβ-Catenin in Bt-474 and Mcf-7 breast carcinoma cells is likely presentin a complex with E-Cadherin, rather than constitutively active in thenucleus. Although, in Mcf-7, it is likely that there are low levels ofWnt dependent transcription, this is clearly not sufficient to have aneffect on the expression of the Wnt target gene Cyclin D1. Knockdown ofPygopus, on the other hand, resulted in a reduction in expression ofCyclin D1, which is a critical regulator of G1 to S phase transition ofthe cell cycle. Therefore, the decrease in numbers of Mcf-7 cells byreduction of Pygopus is possibly a result of effects that cause cellcycle arrest.

Inhibition of β-Catenin by ICAT had no effect on HeLa cell growth,consistent with our observations using β-Catenin siRNA in MCF-7 cells.These data suggest that at least in two cancer cell lines, canonical Wntsignaling does not appear to be important for cell growth. These dataimply an additional role of Pygopus independently of β-Catenin and Wntsignaling.

Limited studies to date outline the role of Pygopus proteins in Wntsignaling and normal development. The function of hPygo in Wnt signalinghas also been addressed in colorectal cancer cells that displayconstitutive Wnt signaling due to identified mutations in APC andβ-Catenin. It is likely, therefore, that Pygopus knockdown, likeβ-Catenin, might also inhibit the growth of colorectal cells.

Our data indicate that the decrease in Mcf-7 cell proliferation may be aresult of cell cycle arrest and that Pygopus may have important rolesindependent of β-Catenin. These results provide new insight into thefunction Pygopus in cancer and suggest that Pygopus is a suitable cancertherapeutic target.

EXAMPLE 3 Pygopus is a Diagnostic and Therapeutic Target in CervicalCancer

FIG. 16 shows knockdown of endogenous hPygo2 using antisense ON in HeLacervical cancer cells. Reagent control (Oligofectamine), antisenseXenopus Pygopus2 (non-specific), and four base mismatch (mismatch)controls are indicated. Knockdown of hPygo2 by antisense ON results in adecrease of HeLa cell numbers 48 and 72 hours after transfection. Cellnumber was assayed for by direct counting with a hemacytometer usingtrypan blue exclusion. RT-PCR analysis shows specific knockdown ofhPygo2 mRNA without effecting expression of the related Pygo familymember, hPygo1. RT− is the negative control, without reversetranscriptase. Western blot analysis shows knockdown of endogenoushPygo2 protein. Levels of cDNA and protein were standardized using GAPDHand beta-Actin. Experiments were performed in triplicate.

EXAMPLE 4 Anti-hPygo2 Antibodies Identify Malignant Tumor Cells inOvarian, Breast and Lung Cancer

Anti-hPygo2 antibodies were used in immuno-histochemical analysis toidentify malignant tumor cells from ovarian epithelial, breast, and lungcancer. Staining of archived tumors, as determined by a licencedpathologist, using anti-hPygo2 antibodies, indicates that Pygopus isspecifically overexpressed in a variety of ovarian epithelial (FIG. 15A,C) tumors and in malignant breast (FIG. 15G) and lung cancer (FIG.15H). Negative staining with pre-immune and secondary antibody alonedemonstrates specificity of the antibody.

1. A method for determining the presence or absence of a cancer in apatient, the method comprising the steps of: (a) determining the levelof Pygopus gene expression in a biological sample obtained from apatient, and (b) comparing the level of Pygopus gene expression in thebiological sample to a predetermined cut-off value, to determine whetherPygopus expression is higher in the biological sample; therefromdetermining the presence or absence of cancer in the patient.
 2. Amethod for monitoring the progression of a cancer in a patient, themethod comprising the steps of: (a) determining the presence or absenceof cancer in the patient according to the method of claim 1; (b)repeating step (a) using a biological sample obtained from the patientat a subsequent time; and (c) comparing the level of Pygopus geneexpression detected in step (b) to the level of Pygopus gene expressiondetected in step (a); and therefrom monitoring the progression of thecancer in the patient.
 3. The method according to claim 1 wherein thepredetermined cut-off value is the level of Pygopus gene expression in anormal biological sample.
 4. The method according to claim 1 wherein thecancer is ovarian cancer, and the biological sample is a tissue biopsycontaining epithelial ovarian cells.
 5. (canceled)
 6. The methodaccording to claim 1 wherein the Pygopus gene is hPygo2 as shown in SEQID NO:
 1. 7. The method according claim 1 wherein the Pygopus gene ishPygo1 as shown in SEQ ID NO:3.
 8. The method according to claim 1wherein the level of Pygopus gene expression is determined by the amountof Pygopus protein.
 9. The method according to claim 1 wherein the levelof Pygopus gene expression is determined by the amount of Pygopus mRNA.10. A kit for determining the presence or absence of a cancer in apatient according to the method of claim 1, the kit comprising a reagentcapable of detecting Pygopus protein or mRNA in a biological sampleobtained from the patient, and instructions for using the reagent todetermine whether the level of Pygopus gene expression in the biologicalsample is higher compared to a predetermined cut-off value, andtherefrom determining the presence or absence of cancer in the patient.11-30. (canceled)
 31. A method for obtaining a compound which inhibitstumor cell proliferation, wherein the tumor cell express Pygopus, themethod comprising: (a) delivering the oligonucleotide of claim 46 intoepithelial ovarian carcinoma or breast cancer cells; and (b) determiningwhether the delivered oligonucleotide inhibits proliferation of thecancer cells. 32-38. (canceled)
 39. A method for inhibiting tumor cellproliferation, the method comprising delivering to the tumor cell aproliferation-inhibiting amount of the oligonucleotide of claim 46 whichreduces expression of a Pygopus-encoding nucleic acid.
 40. The methodaccording to claim 39 wherein the tumor cell is an epithelial ovariancarcinoma cell or breast cancer cell. 41-45. (canceled)
 46. Anoligonucleotide which is an antisense oligonucleotide, a shortinterfering RNA (siRNA) or a siRNA-like molecule, targeted to hPygo2(SEQ ID NO:1) in the region from nucleotide 437 to 1156 of SEQ ID NO:1,wherein said antisense oligonucleotide, siRNA or siRNA-like moleculespecifically hybridizes with said region and reduces the expression ofhPygo2.
 47. An oligonucleotide which is an antisense oligonucleotide, ashort interfering RNA (siRNA) or a siRNA-like molecule, targeted tohPygo1 (SEQ ID NO:3) in the region from nucleotide 253 to 1023 of SEQ IDNO:3, wherein said antisense oligonucleotide, siRNA or siRNA-likemolecule specifically hybridizes with said region and reduces theexpression of hPygo
 1. 48-49. (canceled)
 50. The oligonucleotideaccording to claim 46 having the sequence selected from the groupconsisting of SEQ ID NOS:5-14.
 51. The antisense oligonucleotideaccording to claim 50 having the sequence of SEQ ID NO:9.
 52. Theoligonucleotide according to claim 46 having the sequence selected fromthe group consisting of SEQ ID NOS:15-19.
 53. The oligonucleotideaccording to claim 52 having the sequence of SEQ ID NO: 15 or
 18. 54. Amethod for obtaining a compound which inhibits tumor cell proliferation,wherein the tumor cell express Pygopus, the method comprising: (a)delivering the oligonucleotide of claim 47 into epithelial ovariancarcinoma or breast cancer cells; and (b) determining whether theoligonucleotide inhibits proliferation of the cancer cells.
 55. A methodfor inhibiting tumor cell proliferation, the method comprisingdelivering to the tumor cell a proliferation-inhibiting amount of theoligonucleotide of claim 47 which reduces expression of aPygopus-encoding nucleic acid.
 56. The method according to claim 55wherein the tumor cell is an epithelial ovarian carcinoma cell or breastcancer cell.